Biomarkers for risk assessment and treatment monitoring in heart failure patients guided by natriuretic peptides

ABSTRACT

The present invention relates to a method for identifying a patient who is eligible to an intensification of heart failure therapy. Furthermore, the present invention relates to a method for optimizing BNP-type peptide guided heart failure therapy. The methods are based on the measurement of the level of at least one marker in a sample from a patient who has heart failure and who receives BNP-type peptide guided heart failure therapy. Further envisaged by the present invention are kits and devices adapted to carry out the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/EP2015/051239 filed Jan. 22, 2015, and claims priority toEuropean Patent Application No. 14152777.0 filed Jan. 28, 2014, thedisclosures of which are hereby incorporated by reference in theirentirety.

SUMMARY

The present invention relates to a method for identifying a patient whois eligible to an intensification of heart failure therapy. Furthermore,the present invention relates to a method for optimizing BNP-typepeptide guided heart failure therapy. The methods are based on themeasurement of the level of at least one marker in a sample from apatient who has heart failure and who receives BNP-type peptide guidedheart failure therapy. Further envisaged by the present invention arekits and devices adapted to carry out the present invention.

Heart failure (HF) is among the leading causes of morbidity andmortality in many countries worldwide. Although available treatmentoptions can reduce morbidity and mortality in patients with HF, therelative number of eligible patients receiving these treatments remainsunsatisfactorily low. Furthermore, in patients eligible for treatment,therapy has been primarily guided and adjusted by signs and symptoms ofHF to maximal tolerability of drugs (e.g. by NYHA stages, ACC/AHAstages, or congestion scores).

Measurement of natriuretic peptide markers, such as B-type natriureticpeptide (BNP), or its amino-terminal fragment N-terminal proBNP(NT-proBNP), has emerged as an important tool for the diagnosis and riskstratification of patients with HF. Additionally, there is emergingevidence that NT-proBNP is useful in guiding medical therapy in heart.

NT-proBNP guided HF therapy, however, does not identify all patients atrisk of HF decompensation and of adverse events. Consequently, somepatients remain at risk even though they show favorable response totherapy with regards to their NT-proBNP levels. And thus, not allpatients benefit from intensification of heart failure therapy.

Advantageously, it has been found in the studies underlying the presentinvention that the combination of NT-proBNP or BNP with other markersand clinical parameters can be used for monitoring purposes and as aguide for therapy in addition to current standard-of-care to adjust andtitrate therapy in HF patients (chronic or acute HF afterstabilization). These markers and parameters are Creatinine, BUN (urea),Glucose, HbA1c, hsCRP, Cystatin C, IL-6, Prealbumin, sFLt-1, Uric Acid,GDF-15, sST2, Galectin-3, Endostatin, Mimecan, IGFBP-7, Osteopontin,Sodium, Hemoglobin, and Hematocrit, as well as heart rate and QRSduration. Specifically, addition of these measurements to NT-proBNP orBNP together with current standard-of-care are able to further riskstratify HF patients who are already guided by NT-proBNP but may be inneed for more intensified therapy and closer observation. Thus, thepresent invention optimizes heart failure therapy guidance beyondNT-proBNP by considering combinations of natriuretic peptides with othermarkers and/or clinical parameters.

In particular, it has been found in the studies of the present inventionthat the additional determination of the markers of parameters asreferred to above allows for the identification of a subgroup ofpatients which display a level of a BNP-type peptide which is below thereference level for said BNP-type peptide indicative for theintensification of heart failure therapy, but which nevertheless areeligible to an intensification of heart failure treatment. Thanks to thepresent invention, patients can be identified which require anintensification of heart failure therapy which based on the measurementof the level of a BNP-type alone would have not received an intensifiedheart failure therapy.

Accordingly, the present invention is directed to a method foridentifying or selecting a patient who is eligible to an intensificationof heart failure therapy, said method comprising the steps of

-   -   (a) measuring the level of at least one marker selected from the        group consisting of creatinine, urea, sodium, glucose, HbA1c        (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive        protein, in particular high sensitive CRP), Cystatin C, IL-6        (Interleukin 6), Prealbumin, sFlt-1 (soluble fms-like tyrosine        kinase-1), uric acid, GDF-15 (Growth Differentiation Factor 15),        Galectin-3 (Gal-3), Endostatin, Mimecan, IGFBP7 (Insulin Growth        Factor Binding Protein 7), sST2 (soluble ST2), and Osteopontin        in a sample from a patient who has heart failure and who        receives BNP-type peptide guided heart failure therapy, in        particular NT-proBNP guided heart failure therapy or BNP guided        heart failure therapy, and    -   (b) comparing the level (or levels) of the marker (or markers)        measured in (a) to a reference level (or reference levels).

In an embodiment, the method further comprises step (c) of identifyingor selecting a patient who is eligible to an intensification of heartfailure therapy, i.e. of BNP-type peptide guided therapy.

In addition, the method may comprise step (d) of intensifying heartfailure therapy or recommending intensification of heart failure therapy(if the patient has been identified as to be eligible to intensificationof heart failure therapy). Accordingly, the present invention alsoenvisages a method of intensifying heart failure therapy, said methodcomprising steps (a) to (d) as set forth above.

The method of the present invention, preferably, is an ex vivo or invitro method. Moreover, it may comprise steps in addition to thoseexplicitly mentioned above. For example, further steps may relate tosample pre-treatments or evaluation of the results obtained by themethod. The method may be carried out manually or assisted byautomation. Preferably, step (a) and/or (b) may in total or in part beassisted by automation, e.g., by a suitable robotic and sensoryequipment for the measurement in steps (a) or a computer-implementedidentification in step (c).

In an embodiment the aforementioned method may additionally compriseassessing or providing the QRS duration and the comparison of the thusdetermined QRS duration to a reference.

Further, it is envisaged to assess or to provide the QRS durationinstead of measuring the level of the at least one marker in step a) andto compare the thus determined QRS duration to a reference.

Accordingly, the present invention further envisages a method foridentifying or selecting a patient who is eligible to an intensificationof heart failure therapy, said method comprising the steps of

-   -   (a) assessing or providing the QRS duration of a patient who has        heart failure and who receives BNP-type peptide guided heart        failure therapy, and    -   (b) comparing QRS duration to a reference,        wherein a QRS duration which is increased as compared to the        reference is indicative for a patient who is eligible to        intensification of heart failure therapy, whereas a QRS duration        which is decreased as compared to the reference is indicative        for a patient who is not eligible to intensification of heart        failure therapy

The “patient” as referred to herein is, preferably, a mammal. Mammalsinclude, but are not limited to, domesticated animals (e.g., cows,sheep, cats, dogs, and horses), primates (e.g., humans and non-humanprimates such as monkeys), rabbits, and rodents (e.g., mice and rats).In certain embodiments, the patient is a human patient. The terms“subject” and “patient” are used interchangeably herein.

The phrase “selecting a patient” or “identifying a patient” as usedherein refers to using the information or data generated relating to thelevel of the at least one marker as referred to in the context of thepresent invention in a sample of a patient to identify or selecting thepatient as more likely to benefit or less likely to benefit from anintensification of heart failure therapy. Preferably, a subject who iseligible to said intensification requires said intensification, whereasa subject who is not eligible to said intensification does not requiresaid intensification.

It is to be understood that a subject who is eligible to intensificationof heart failure therapy will benefit from the intensification, whereasa subject who is not eligible to said intensification may not benefitfrom said intensification, e.g. may experience adverse side-effects orharm from the intensification. In particular, a subject benefits fromthe intensification, if the intensification reduces the risk ofmortality of said subject and/or reduces the risk of hospitalizationand/or of cardiac decompensation of said subject, in particular within awindow period of 18 months or 3 years after the sample has beenobtained. Preferably, the aforementioned risk (or risks) is (are)reduced by 5%, more preferably by 10%, even more preferably by 15% and,most preferably by 20%. Preferably, the hospitalization and mortalityreferred to herein shall be due to heart failure.

In contrast, a subject who is not eligible to intensification of heartfailure therapy will not benefit (in particular will not benefitsignificantly) from the intensification. In particular, a subject doesnot benefit from the intensification, if the intensification does notreduce (in particular, does not reduce significantly) the risk ofmortality of said subject and/or does not reduce (in particular, doesnot reduce significantly) the risk of hospitalization and/or of cardiacdecompensation of said subject and/or increases the risk of unwantedside effects, in particular within a window period of 18 months or 3years after the sample has been obtained. In this case, unnecessaryhealth care costs can be avoided, if the therapy is not intensified.Further, adverse side effects that may result from the intensificationcan be avoided.

Thus, by identifying a subject who is eligible to intensification ofheart failure therapy, it can be assessed whether said subject willbenefit from the intensification of heart failure therapy, or not.Accordingly, the present invention also relates to a method ofidentifying a subject who will benefit from intensification of heartfailure therapy, based on the steps set forth herein elsewhere.

The information or data used or generated may be in any form, written,oral or electronic. In some embodiments, using the information or datagenerated includes communicating, presenting, reporting, storing,sending, transferring, supplying, transmitting, dispensing, orcombinations thereof. In some embodiments, communicating, presenting,reporting, storing, sending, transferring, supplying, transmitting,dispensing, or combinations thereof are performed by a computing device,analyzer unit or combination thereof. In some further embodiments,communicating, presenting, reporting, storing, sending, transferring,supplying, transmitting, dispensing, or combinations thereof areperformed by a laboratory or medical professional. In some embodiments,the information or data includes a comparison of the level (levels) ofthe at least one marker to a reference level (or to reference levels).

As described herein below in more detail, a subject who is eligible tointensification of heart failure treatment, shall be also monitored atshort intervals, whereas a subject who is not eligible tointensification of heart failure treatment (i.e. who does not requireintensification of heart failure treatment) shall be monitored at longintervals. Therefore, in addition to the decision whether heart failuretreatment shall be intensified or not, it can be assessed whether thesubject shall be monitored at short intervals or long intervals.

DETAILED DESCRIPTION

As will be understood by those skilled in the art, the assessment madeby the method of the present invention is usually not intended to becorrect for 100% of the subjects to be diagnosed. The term, however,requires that the assessment is correct for a statistically significantportion of the subjects (e.g. a cohort in a cohort study). Whether aportion is statistically significant can be determined without furtherado by the person skilled in the art using various well known statisticevaluation tools, e.g., determination of confidence intervals, p-valuedetermination, Student's t-test, Mann-Whitney test etc. Details arefound in Dowdy and Wearden, Statistics for Research, John Wiley & Sons,New York 1983. Preferred confidence intervals are at least 90%, at least95%, at least 97%, at least 98% or at least 99%. The p-values are,preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001.

It is envisaged in the context of the present invention that the subjectsuffers from heart failure (HF), in particular from chronic heartfailure. Further, the subject may suffer from stabilized acute heartfailure.

The term “heart failure” as used herein relates to a diastolicdysfunction or, in particular, of a systolic dysfunction of the heartbeing accompanied by overt signs of heart failure as known to the personskilled in the art. Preferably, heart failure referred to herein ischronic heart failure (which preferably is caused by systolicdysfunction). Heart failure according to the present invention includesovert and/or advanced heart failure. In overt heart failure, the patientshows symptoms of heart failure as known to the person skilled in theart.

HF can be classified into various degrees of severity.

According to the NYHA (New York Heart Association) classification, heartfailure patients are classified as belonging to NYHA classes I, II, IIIand IV. A patient having heart failure has already experiencedstructural and functional changes to his pericardium, myocardium,coronary circulation or cardiac valves. He will not be able to fullyrestore his health, and is in need of a therapeutical treatment.Patients of NYHA Class I have no obvious symptoms of cardiovasculardisease but already have objective evidence of functional impairment.Patients of NYHA class II have slight limitation of physical activity.Patients of NYHA class III show a marked limitation of physicalactivity. Patients of NYHA class IV are unable to carry out any physicalactivity without discomfort. They show symptoms of cardiac insufficiencyat rest.

This functional classification is supplemented by the more recentclassification by the American College of Cardiology and the AmericanHeart Association (see J. Am. Coll. Cardiol. 2001; 38; 2101-2113,updated in 2005, see J. Am. Coll. Cardiol. 2005; 46; e1-e82). 4 stagesA, B, C and D are defined. Stages A and B are not HF but are consideredto help identify patients early before developing “truly” HF. Stages Aand B patients are best defined as those with risk factors for thedevelopment of HF. For example, patients with coronary artery disease,hypertension, or diabetes mellitus who do not yet demonstrate impairedleft ventricular (LV) function, hypertrophy, or geometric chamberdistortion would be considered stage A, whereas patients who areasymptomatic but demonstrate LV hypertrophy and/or impaired LV functionwould be designated as stage B. Stage C then denotes patients withcurrent or past symptoms of HF associated with underlying structuralheart disease (the bulk of patients with HF), and stage D designatespatients with truly refractory HF.

As used herein, the term “heart failure”, in particular, refers tostages B and C of the ACC/AHA classification referred to above. In thesestages, the subject shows typical symptoms of heart failure.Accordingly, the patient, preferably, has heart failure classified asstage B or C according to the ACC/AHA classification. Also preferably,the patient has heart failure according to class II or III of the NYHAclassification.

Preferably, the heart failure is due to impaired systolic function.Accordingly, it is, in particular, envisaged that the patient suffersfrom systolic heart failure. Preferably, the patient has a leftventricular ejection fraction (LVEF) of less 50%, more preferably, ofless than 45%, and most preferably, of less than 40%.

The patient to be tested on accordance with the method of the presentinvention shall receive BNP-type peptide guided therapy, i.e. BNP-typepeptide guided heart failure therapy. The terms “BNP-type peptide guidedtherapy” and “BNP-type peptide guided heart failure therapy” are wellknown in the art. Accordingly, the patient to the tested shall receiveheart failure therapy (to be more precise at the time at which thesample is obtained) which is guided by a BNP-type peptide. Thus, it isenvisaged that at least one decision as regards to the heart failuretherapy for said patient may have been made in the past (and thus priorbefore obtaining the sample to be tested) based on the level of aBNP-type peptide in said patient, in particular based on the blood,serum or plasma level of a BNP-type peptide in said patient.Accordingly, the patient's level of a BNP-type peptide may have beenconsidered for past decisions on heart failure treatment. Further, it isenvisaged that the present decision with respect to heart failuretherapy is the first decision which involves the consideration of thelevel of a BNP-type peptide. Accordingly, the patient who receivesBNP-type peptide guided heart failure therapy may be a patient in whichBNP-type peptide guided heart failure therapy is initiated (inparticular immediately after the sample to be tested has been obtained).Nevertheless, said patient may have received heart failure therapypreviously which has not been guided by a BNP-type peptide.

Preferred BNP-type peptides are disclosed elsewhere herein. The BNP-typepeptide guided therapy, preferably, may be BNP (Brain natriureticpeptide) guided therapy or, in particular, NT-proBNP (N-terminal probrain natriuretic peptide) guided therapy (for an explanation of thesemarkers, see elsewhere).

In BNP-type peptide guided therapy, the level of a BNP-type peptide isused for managing heart failure treatment. Based on the level, decisionson the heart failure treatment are made. In principle, a patient with anincreased level of a BNP-type peptide receives a more intensifiedtherapy than a patient with a reduced level of this marker. BNP-typepeptide guided therapy is well known in the art and is e.g. described bySanders-van Wijk et al. Eur J Heart Fail (2013) 15 (8): 910-918.Further, BNP-type peptide guided therapies are reviewed by Januzzi, seeArchives of Cardiovascular disease (2012), 105, 40 to 50. Bothreferences are herewith incorporated by reference with respect to theirentire disclosure content.

In a preferred embodiment, the patient displays a level (in particular ablood, serum or plasma level) of a BNP-type peptide which is below thereference level for said BNP-type peptide being indicative ofintensification of heart failure therapy. Accordingly, the patient shallbe a patient having a level of a BNP-type peptide which would, whentaken alone (i.e. not in combination with the at least one furthermarker as set forth in step (a) of the aforementioned method, beindicative for a patient who is not eligible to an intensification ofheart failure therapy. Preferred reference levels for said BNP-typepeptide being indicative of intensification of heart failure therapy tobe applied in the context of the present invention are those describedin the Examples. Preferred reference levels are within a range fromabout 80 to 400 pg/ml, or, in particular, from about 80 to 200 pg/ml forBNP, or within a range from about 450 to 2200 pg/ml, or in particularfrom about 800 pg/ml to 1200 pg/ml for NT-proBNP. Further preferredreference levels are about 100 pg/ml or 400 pg/ml for BNP, and about1000 pg/ml, or 1200 pg/ml for NT-proBNP. Thus, the patient in accordancewith the present invention may display a level of NT-proBNP, inparticular a blood, serum or plasma level of NT-proBNP, of less than1000 pg/ml or 1200 pg/ml.

Further, it is envisaged that the patient who displays a level of aBNP-type which is below the reference level for said BNP-type peptidebeing indicative of intensification of heart failure therapy has a level(in particular a blood, serum or plasma level) of BNP within the rangefrom about 80 to about 400 pg/ml, in particular within the range ofabout 80 to about 200 pg/ml. Also, the patient who displays a level of aBNP-type which is below the reference level for said BNP-type peptidebeing indicative of intensification of heart failure therapy may have alevel (in particular a blood, serum or plasma level) of NT-proBNP withinthe range of 450 to 2200 pg/ml, in particular within the range of 800 to1200 pg/ml.

Preferably, the term “about” as used herein encompasses a range of + and−20%, more preferably a range of + and −10%, even more preferably arange of + and −5%, and most preferably a range of + and −2%, relativeto the specific amount, e.g., indication of a an amount of “about 100”is meant to encompass an amount within a range from 80 to 120. Also, theterm “about” refers to the exact amount. Preferably, the levels aremeasured as described in the Examples.

The term “heart failure therapy” (herein also referred to as “heartfailure treatment”) as used herein, preferably, refers to any treatmentthat allows for treating heart failure. Preferably, the term encompasseslife style changes, diet regimen, interventions on the body as well asadministration of appropriate medicaments, use of devices and/or organtransplants for the treatment of the patient suffering from heartfailure.

Life style changes include smoking cessation, reduction of alcoholconsumption, increased physical activity, weight loss, sodium (salt)restriction, weight management and healthy eating (such as daily fishoil).

Preferred devices to be applied are pacemakers and resynchronizationdevices, defibrillator, intra-aortic balloon pumps, and left ventricularassist devices.

In a preferred embodiment, the heart failure therapy is medicinal heartfailure therapy. Accordingly, the heart failure therapy preferablyencompasses administration of one ore more medicaments. The tem“administering” as used herein is used in the broadest sense and interalia encompasses oral, enteral, topical administration and “parenteraladministration”. “Parenteral administration” and “administeredparenterally” as used herein mean modes of administration other thanenteral and topical administration, usually by injection, and include,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural,intrasternal injection and infusion. In an embodiment, the medication isadministered orally.

Medicaments suitable for the treatment of heart failure are well knownin the art, see e.g. Heart Disease, 2008, 8th Edition, Eds. Braunwald,Elsevier Sounders, chapter 24 or the ESC Guidelines for the diagnosisand treatment of acute and chronic heart failure (European Heart Journal(2008) 29, 2388-2442). Preferably, the heart failure treatment includesadministration of at least one medicament selected from the groupconsisting of angiotensin converting enzyme inhibitors (ACE inhibitors),angiotensin II receptor blockers (frequently also referred to asangiotensin II receptor antagonists), beta adrenergic blockers (hereinalso referred to as beta blockers), diuretics, aldosterone antagonists,adrenergic agonists, positive inotropic agents, calcium antagonists,hydralazine, nitrates, and aspirin. It is particularly preferred thatthe medicament is an angiotensin converting enzyme inhibitor, anangiotensin II receptor blocker, a beta blocker and/or an aldosteroneblocking agent.

Preferred ACE-inhibitors include benazepril, captopril, cilazapril,enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril,ramipril, spirapril, and trandolapril. A particularly preferredinhibitor is enalapril.

Preferred beta blockers include cebutolol, alprenolol, atenolol,betaxolol, bisoprolol, bupranolol, carazolol, carteolol, carvedilol,celiprolol, metipranolol, metoprolol, nadolol, nebivolol, oxprenolol,penbutolol, pindolol, propanolol, sotalol, tanilolol, and timolol. Aparticularly preferred beta blocker is atenolol, bisoprolol, carvedilol,or metoprol.

Preferred angiotensin II receptor antagonists are Losartan, Valsartan,Irbesartan, Candesartan, Telmisartan, and Eprosartan. A particularlypreferred antagonist is Losartan or Valsartan.

Preferred diuretics are loop diuretics, thiazide and thiazide-likediuretics, K-sparing diuretics, mineralocorticoid receptor antagonists,and vasopres sin antagonists.

Preferred aldosterone antagonists are Eplerone, Spironolactone,Canrenone, Mexrenone, Prorenone; and statines, in particularAtorvastatin, Fluvastatin, Lovastatin, Pravastatin, Rosuvastatin, andSimvastatin. A particularly preferred antagonist is Spironolactone.

Preferred positive inotropic agents are digoxin and digitoxin.

Preferred calcium antagonists are dihydropyridines, verapamil, anddiltiazem.

Preferred adrenergic agonists are dobutamine, dopamine, epinephrine,isoprotenerol, nore-pinephrine, and phenylephrine.

The heart failure therapy to be intensified, or not, can be anytreatment as set forth herein above. In a preferred embodiment, however,the heart failure therapy comprises administration of at least onemedicament as set forth above. In an even more preferred embodiment, theheart failure therapy comprises administration of at least onemedicament selected from the group consisting of an angiotensinconverting enzyme inhibitor, an angiotensin II receptor blocker, a betablocker, a diuretic, and an aldosterone antagonist. Most preferably, theheart failure treatment to be intensified comprises the combinedadministration of a beta blocker and an ACE inhibitor.

In accordance with the method of the present invention, it shall beassessed whether heart failure treatment of the patient to be testedshall be intensified, or not. Preferably, the intensification of heartfailure treatment comprises at least one of the following:

-   -   increasing the dosage of a previously administered medicament or        of previously administered medicaments,    -   the administration of a further or another medicament (or        medicaments), in particular the administration of a further        medicament (or medicaments) having a different mode of action        that the previously administered medicament(s),    -   device therapy, in particular use of pacemaker devices, cardiac        resynchronization therapy (CRT), implantable defibrillator        devices (ICD) or left ventricular assist devices (LVAD), life        style changes, and    -   combinations thereof.

Preferably, the intensification comprises increasing the dosage of apreviously administered medicament or of previously administeredmedicaments, in particular increasing the dosage of a medicamentselected from the group consisting of a diuretic, an angiotensinconverting enzyme inhibitor, an angiotensin II receptor blocker, analdosterone antagonist, and a beta blocker. How to increase the dosage,it well known in the art, and, e.g., may be derived from the guidelines.Preferably, the dosing of these medicaments may be increased until themaximally recommended therapeutic dose or until the maximally tolerateddose, whatever is reached first. Also preferably, the dosage may beincreased by at least 30% or at least 50%.

Also preferably, the intensification comprises the administration of afurther medicament (or medicaments), in particular the administration ofa further medicament (or medicaments) having a different mode of actionthan the previously administered medicaments, or the application offurther devices (i.e. of medicaments/devices that were notadministered/used prior to carrying out the method of the presentinvention). Preferred further medicaments include hydralazine, nitrates,inotropic agents, and adrenergic agents. Preferred devices includepacemaker devices, cardiac resynchronization therapy (CRT), andimplantable defibrillator devices (ICD).

Also, the intensification of heart failure treatment may furtherencompass monitoring the patient at short intervals. Accordingly, bycarrying out the method of the present invention a patient can beidentified who requires closer monitoring, in particular with respect tothe heart failure therapy (and, thus, closer observation). With “closermonitoring” it is, preferably, meant that the levels of the markers asreferred herein in connection with the method of the present inventionare measured in at least one further sample obtained from the patientafter a short interval after the sample referred to in step a) of themethod of the present invention. Preferred short intervals are mentionedherein below.

A patient who does not require intensification of heart failuretreatment, preferably, can continue the heart failure treatment withoutchanging the treatment regimen. Thus, it is not necessary to adapt thedosage of the administered medicament(s) and/or to change themedicaments.

The term “sample” refers to a sample of a body fluid, to a sample ofseparated cells or to a sample from a tissue or an organ. Samples ofbody fluids can be obtained by well-known techniques and include,samples of blood, plasma, serum, urine, lymphatic fluid, sputum,ascites, bronchial lavage or any other bodily secretion or derivativethereof. Tissue or organ samples may be obtained from any tissue ororgan by, e.g., biopsy. Separated cells may be obtained from the bodyfluids or the tissues or organs by separating techniques such ascentrifugation or cell sorting. E.g., cell-, tissue- or organ samplesmay be obtained from those cells, tissues or organs which express orproduce the biomarker. The sample may be frozen, fresh, fixed (e.g.formalin fixed), centrifuged, and/or embedded (e.g. paraffin embedded),etc. The cell sample can, of course, be subjected to a variety ofwell-known post-collection preparative and storage techniques (e.g.,nucleic acid and/or protein extraction, fixation, storage, freezing,ultrafiltration, concentration, evaporation, centrifugation, etc.) priorto assessing the amount of the marker in the sample. Likewise, biopsiesmay also be subjected to post-collection preparative and storagetechniques, e.g., fixation.

In an embodiment the sample is a blood, serum or, in particular, aplasma sample.

The sample may be obtained from the patient in increasing order ofpreference at least one month, at least six months, or at least 12months after initiation of heart failure therapy, in particular ofBNP-type peptide guided therapy. Preferably, said therapy is medicinalheart failure therapy.

The level of the biomarkers as referred to herein can be determined inthe same sample or in different samples (i.e. in two or three differentsamples) from the patient.

The term “measuring” the level of a marker as referred to herein refersto the quantification of the biomarker, e.g. to determining the level ofthe biomarker in the sample, employing appropriate methods of detectiondescribed elsewhere herein. In an embodiment, the level of the at leastone biomarker is measured by contacting the sample with a detectionagent that specifically binds to the respective marker, thereby forminga complex between the agent and said marker, detecting the level ofcomplex formed, and thereby measuring the level of said marker. If thebiomarker is uric acid, the level of said biomarker may be measured bycontacting the sample with detection agent, in particular an enzyme orcompound, that allows for the conversion of said biomarker, e.g. thatallows for the oxidation of uric acid. The enzyme may be an uricase (EC1.7.3.3) which catalyzes the oxidation of uric acid to5-hydroxyisourate. Also, the enzyme can be a peroxidase. The compoundmay be phosphotungstic acid. If the marker is urea, the detection agentmay be urease. If the marker is glucose, the detection agent may be ahexokinase. If the marker is creatinine, the detection agent may bepicric acid (which forms a complex with creatinine). The level of thecomplex of picric acid and creatinine may be measured.

The term “Growth-Differentiation Factor-15” or “GDF-15” relates to apolypeptide being a member of the transforming growth factor (TGF)cytokine superfamily. The terms polypeptide, peptide and protein areused interchangeable throughout this specification. GDF-15 wasoriginally cloned as macrophage-inhibitory cytokine 1 and later alsoidentified as placental transforming growth factor-15, placental bonemorphogenetic protein, non-steroidal anti-inflammatory drug-activatedgene 1, and prostate-derived factor (Bootcov loc cit; Hromas, 1997Biochim Biophys Acta 1354:40-44; Lawton 1997, Gene 203:17-26;Yokoyama-Kobayashi 1997, J Biochem (Tokyo), 122:622-626; Paralkar 1998,J Biol Chem 273:13760-13767). Similar to other TGF-related cytokines,GDF-15 is synthesized as an inactive precursor protein, which undergoesdisulfide-linked homodimerization. Upon proteolytic cleavage of the Nterminal pro-peptide, GDF-15 is secreted as a ˜28 kDa dimeric protein(Bauskin 2000, Embo J 19:2212-2220). Amino acid sequences for GDF-15 aredisclosed in WO99/06445, WO00/70051, WO2005/113585, Bottner 1999, Gene237: 105-111, Bootcov loc. cit, Tan loc. cit., Baek 2001, Mol Pharmacol59: 901-908, Hromas loc cit, Paralkar loc cit, Morrish 1996, Placenta17:431-441 or Yokoyama-Kobayashi loc cit. GDF-15 as used hereinencompasses also variants of the aforementioned specific GDF-15polypeptides. Such variants have at least the same essential biologicaland immunological properties as the specific GDF-15 polypeptides. Inparticular, they share the same essential biological and immunologicalproperties if they are detectable by the same specific assays referredto in this specification, e.g., by ELISA assays using polyclonal ormonoclonal antibodies specifically recognizing the said GDF-15polypeptides. A preferred assay is described in the accompanyingExamples. Moreover, it is to be understood that a variant as referred toin accordance with the present invention shall have an amino acidsequence which differs due to at least one amino acid substitution,deletion and/or addition wherein the amino acid sequence of the variantis still, preferably, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 85%, at least about 90%,at least about 92%, at least about 95%, at least about 97%, at leastabout 98%, or at least about 99% identical with the amino sequence ofthe specific GDF-15 polypeptides, preferably with the amino acidsequence of human GDF-15, more preferably over the entire length of thespecific GDF-15, e.g. of human GDF-15. The degree of identity betweentwo amino acid sequences can be determined as described above. Variantsreferred to above may be allelic variants or any other species specifichomologs, paralogs, or orthologs. Moreover, the variants referred toherein include fragments of the specific GDF-15 polypeptides or theaforementioned types of variants as long as these fragments have theessential immunological and biological properties as referred to above.Such fragments may be, e.g., degradation products of the GDF-15polypeptides. Further included are variants which differ due toposttranslational modifications such as phosphorylation ormyristylation.

The Insulin like growth factor binding protein (IGFBP) system plays animportant role in cell growth and differentiation. It comprises twoligands, IGF-I and IGF-II, two receptors, type 1 and type 2 IGFreceptors, and as of 1995 six IGF-binding proteins (IGFBPs), IGFBP-1 to-6 (Jones, J. I., et al., Endocr. Rev. 16 (1995) 3-34). Recently theIGFBP family has been expanded to include the IGFBP-related proteins(IGFBP-rPs), which have significant structural similarities with theIGFBPs (Hwa, V., et al., Endocr. Rev 20 (1999) 761-787). Thus, the IGFBPsuperfamily includes the six conventional IGFBPs, which have highaffinity for IGFs, and at least 10 IGFBP-rPs, which not only share theconserved amino-terminal domain of the IGFBPs but also show some degreeof affinity for IGFs and insulin. The IGFBP-rPs are a group ofcysteine-rich proteins that control diverse cellular functions, such ascellular growth, cell adhesion and migration, and synthesis of theextracellular matrix. In addition, these proteins might be involved inbiological processes like tissue proliferation and differentiation,reproduction, angiogenesis, wound repair, inflammation, fibrosis, andtumorigenesis (Hwa, V., et al., Endocr. Rev 20 (1999)761-787).

IGF binding protein 7 (=IGFBP7) is a 30-kDa modular glycoprotein knownto be secreted by endothelial cells, vascular smooth muscle cells,fibroblasts, and epithelial cells (Ono, Y., et al., Biochem Biophys ResComm 202 (1994) 1490-1496). In the literature this molecule has alsobeen denominated as FSTL2; IBP 7; IGF binding protein related protein I;IGFBP 7; IGFBP 7v; IGFBP rP1; IGFBP7; IGFBPRP1; insulin like growthfactor binding protein 7; insulin like growth factor binding protein 7precursor; MAC25; MAC25 protein; PGI2 stimulating factor; and PSF orProstacyclin stimulating factor. Northern blot studies revealed a wideexpression of this gene in human tissues, including heart, brain,placenta, liver, skeletal muscle, and pancreas (Oh, Y., et al., J. Biol.Chem. 271 (1996) 30322-30325).

IGFBP7 was initially identified as a gene differentially expressed innormal leptomeningeal and mammary epithelial cells, compared with theircounterpart tumor cells, and named meningioma-associated cDNA (MAC25)(Burger, A. M., et al., Oncogene 16 (1998) 2459-2467). The expressedprotein was independently purified as a tumor derived adhesion factor(later renamed angiomodulin) (Sprenger, C. C., et al., Cancer Res 59(1999) 2370-2375) and as a prostacyclin stimulating factor (Akaogi, K.,et al., Proc Natl Acad Sci USA 93 (1996) 8384-8389). It has additionallybeen reported as T1A12, a gene down-regulated in breast carcinomas(StCroix, B., et al., Science 289 (2000) 1197-1202).

Differential expression of IGFBP7 mRNA was measured in patientssuffering from various diseases including cardiac disease, kidneydisease, inflammatory diseases (U.S. Pat. No. 6,709,855 to Scios Inc.)and vascular graft disease (US 2006/0,003,338).

A number of different assays has been described and used to test for thehormone binding properties of IGFBP7. Low affinity IGF binding wasanalyzed via competitive affinity cross-linking assays. Recombinanthuman mac25 protein specifically binds IGF-I and -II (Oh, Y., et al., J.Biol. Chem. 271 (1996) 20322-20325; Kim, H. S., et al., Proc. Natl.Acad. Sci USA 94 (1997) 12981-12986.) IGFBP activity can also bedetected by measuring the ability of the protein to bind radiolabeledIGF in Western ligand blotting.

Preferably, the term “IGFBP7” refers to human IGFBP7. The sequence ofthe protein is well known in the art and is e.g. accessible via GenBank(NP_001240764.1). IGFBP7 as used herein, preferably, encompasses alsovariants of the specific IGFBP7 polypeptides. For an explanation of theterm “variants”, please see above.

Immunological determination of circulating IGFBP7 was performedrecently. Low levels of this analyte were detected in random human seraand increased serum levels have been seen in association withinsulin-resistance (Lopez-Bermejo, A., et al., J. Clinical Endocrinologyand Metabolism 88 (2003) 3401-3408, Lopez-Bermejo, A., et al., Diabetes55 (2006) 2333-2339).

The marker Endostatin is well known in the art. Endostatin wasoriginally isolated from murine hemangioendothelioma as a 20 kDAproteolytic fragment of type XVIII collagen (O'Reilly, M. S. et al.,Cell 88 (1997) 277-285). Collagens represent a family of extracellularmatrix proteins with a characteristic triple-helical conformationforming supra-molecular aggregates that play a dominant role inmaintaining tissue structural integrity. Excessive collagen depositionleads to fibrosis disrupting the normal functioning of surroundingtissues. Collagen XVIII is a member of the Multiplexin family ofcollagens with multiple interruptions in the central triple-helicaldomain and a unique non-triple-helical domain at the C-terminus mainlyin basement membranes. The sequence of the short isoform of human typealpha 1-chain of collagen XVIII (SwissProt: P39060) is e.g. disclosed inWO2010/124821 which herewith is incorporated by reference with respectto its entire disclosure content.

Endostatin is released from the alpha 1 chain of collagen XVIII byaction of various proteolytic enzymes (for details see Ortega, N. andWerb, Z., Journal of Cell Science 115 (2002) 4201-4214—the fulldisclosure of this paper is herewith incorporated by reference).Endostatin as used herein is represented by the collagen XVIII fragmentspanning from amino acid position 1337 to amino acid position 1519 ofcollagen XVIII as disclosed in WO2010/124821. The hinge region at theC-terminus of the alpha chain of collagen XVIII contains severalprotease sensitive sites and a number of enzymes, including neutrophilelastase, cathepsins and matrix metalloproteinases are known to generateendostatin by cleaving the collagen chain in this region. Theseproteases do not exclusively release endostatin but also may releaseother, larger fragments that contain the endostatin sequence. As obviousto the skilled artisan such larger fragments will also be measured by animmunoassay for endostatin.

Osteopontin (OPN), also known as bone sialoprotein I (BSP-1 or BNSP),early T-lymphocyte activation (ETA-1), secreted phosphoprotein 1 (SPP1),2ar and Rickettsia resistance (Ric), is a polypeptide which is a highlynegatively charged, extracellular matrix protein that lacks an extensivesecondary structure. It is composed of about 300 amino acids (297 inmouse; 314 in human) and is expressed as a 33-kDa nascent protein; thereare also functionally important cleavage sites. OPN can go throughposttranslational modifications which increase its apparent molecularweight to about 44 kDa. The sequence of osteopontin is well known in theart (human osteopontin: UniProt P10451, GenBank NP_000573.1) Osteopontinis found in normal plasma, urine, milk and bile (U.S. Pat. No.6,414,219; U.S. Pat. No. 5,695,761; Denhardt, D. T. and Guo, X., FASEBJ. 7 (1993) 1475-1482; Oldberg, A., et al., PNAS 83 (1986) 8819-8823;Oldberg, A., et al., J. Biol. Chem. 263 (1988) 19433-19436; Giachelli, CM., et al., Trends Cardiovasc. Med. 5 (1995) 88-95). The human OPNprotein and cDNA have been isolated and sequenced (Kiefer M. C, et al.,Nucl. Acids Res. 17 (1989) 3306). OPN functions in cell adhesion,chemotaxis, macrophage-directed interleukin-10. OPN is known to interactwith a number of integrin receptors. Increased OPN expression has beenreported in a number of human cancers, and its cognate receptors (av-b3,av-b5, and av-b1 integrins and CD44) have been identified. In vitrostudies by Irby, R. B., et al., Clin. Exp. Metastasis 21 (2004) 515-523indicate that both endogenous OPN expression (via stable transfection)as well as exogenous OPN (added to culture medium) enhanced the motilityand invasive capacity of human colon cancer cells in vitro.

Endostatin is a potent inhibitor of angiogenesis and blood vesselgrowth. The relationship between endostatin and cytokine networks isundetermined, but it is known that endostatin is able to alterexpression of a wide range of genes (Abdollahi, A. et al., MoI. Cell 13(2004) 649-663).

Endostatin as used herein, preferably, encompasses also variants of thespecific endostatin polypeptides. For an explanation of the term“variants”, please see above.

Mimecan is a small proteoglycan with leucin-rich repeats and a precursorcomprising 298 amino acids. Other names of mimecan are OGN, osteoglycin,OG, OIF, SLRR3A.

Mimecan is a member of the secreted small leucine rich proteoglycans(SLRP) family with structurally related core proteins. The commonfeature shared by all SLRPs is the tandem leucine-rich repeat (LRR)units in the C-terminal half of the core protein. In the N-terminalregion, however, each class of SLRP has a unique domain containing acysteine cluster with conserved spacing called the LRR N-domain. ClassIII SLRPs contain six carboxyl LRRs and include mimecan, epiphycan andopticin.

Functional studies from mouse knockouts for class I and II members, suchas decorin, biglycan, lumecan and fibromodulin, showed that theSLRP-deficient mice displayed a wide array of defects attributable toabnormal collagen fibrillogenesis suggesting that these SLRPs playimportant roles in establishing and maintaining the collagen matrix(Ameye, L. and Young, M. F., Glycobiology 12 (2002) 107R-116R).Deficiency of class III mimecan also caused collagen fibrilabnormalities (Tasheva, E. S. et al., MoI. Vis. 8 (2002) 407-415).

Mimecan is a multifunctional component of the extracellular matrix. Itbinds to a variety of other proteins (IGF2, IKBKG, IFNB1, INSR, CHUK,IKBKB, NFKBIA, ILl 5, Cd3, retinoic acid, APP, TNF, lipopolysaccharide,c-ab1 oncogene 1, receptor tyrosine kinase, v-src sarcoma viraloncogene). These diverse binding activities may account for the abilityof mimecan to exert diverse functions in many tissues.

Mimecan has been found in cornea, bone, skin and further tissues. Itsexpression pattern is altered in different pathological conditions.Despite the increasing amount of data on the biological role of mimecanits function is still not clear. Mimecan has been shown to be involvedin regulating collagen fibrillogenesis, a process essential indevelopment, tissue repair, and metastasis (Tasheva et al., MoI. Vis. 8(2002) 407-415). It plays a role in bone formation in conjunction withTGF-beta-1 or TGF-beta-2.

The sequence of the human mimecan polypeptide is well known in the artand may be assessed, e.g., via GenBank accession number NP_054776.1GI:7661704. Further, the sequence is disclosed in WO2011/012268. Mimecanas used herein, preferably, encompasses also variants of the specificmimecan polypeptides. For an explanation of the term “variants”, pleasesee above. In context of the present invention, mimecan is preferablydetermined as described in WO2011/012268.

The term “soluble Flt-1” or “sFlt-1” as used herein refers topolypeptide which is a soluble form of the VEGF receptor Flt1. It wasidentified in conditioned culture medium of human umbilical veinendothelial cells. The endogenous soluble Flt1 (sFlt1) receptor ischromatographically and immunologically similar to recombinant humansFlt1 and binds [1251] VEGF with a comparable high affinity. Human sFlt1is shown to form a VEGF-stabilized complex with the extracellular domainof KDR/Flk-1 in vitro. Preferably, sFlt1 refers to human sFlt1. Morepreferably, human sFlt1 can be deduced from the amino acid sequence ofFlt-1 as shown in Genbank accession number P17948, GI: 125361. An aminoacid sequence for mouse sFlt1 is shown in Genbank accession numberBAA24499.1, GI: 2809071.

The term “sFlt-1” used herein also encompasses variants of theaforementioned specific sFlt-1 polypeptide. Such variants have at leastthe same essential biological and immunological properties as thespecific sFlt-1 polypeptide. In particular, they share the sameessential biological and immunological properties if they are detectableby the same specific assays referred to in this specification, e.g., byELISA assays using polyclonal or monoclonal antibodies specificallyrecognizing the said sFlt-1 polypeptide. For a more detailed explanationof the term “variants”, please see above.

Galectin-3 (Gal-3) is a structurally unique member of a family ofbeta-galactoside-binding lectins. Expression of galectin-3 has beenassociated with the epithelium and inflammatory cells includingmacrophages, neutrophils and mast cells. Galectin-3 has been implicatedin a variety of biological processes important in heart failureincluding myofibroblast proliferation, fibrogenesis, tissue repair,cardiac remodeling and inflammation. Galectin-3 is approximately 30 kDaand, like all galectins, contains a carbohydrate-recognition-bindingdomain (CRD) of about 130 amino acids that enable the specific bindingof β-galactosides. Galectin-3 is encoded by a single gene, LGALS3. Itcomprises an N-terminal domain with tandem repeats of short amino acidsegments (a total of 110-130 amino acids) linked to a single C-terminalCRD of about 130 amino acids. It is expressed in the nucleus, cytoplasm,mitochondrion, cell surface, and extracellular space—This protein hasbeen shown to be involved in the following biological processes: celladhesion, cell activation and chemoattraction, cell growth anddifferentiation, cell cycle, and apoptosis. Elevated levels ofgalectin-3 have been found to be significantly associated with higherrisk of death in both acute decompensated heart failure and chronicheart failure populations (see, e.g., DeFilippi C, Christenson R, ShahR, et al. (2009). Clinical validation of a novel assay for galectin-3for risk assessment in acutely destabilized heart failure.)

The protein sequence of Galectin-3 is well known in the art, see e.g.uniprot accession number P17931 (version 5, Nov. 25, 2008), GenBankaccession number NP_002297.2 NM_002306.3.

ST2 is a member of the IL-1 receptor family that is produced by cardiacfibroblasts and cardiomyocytes under conditions of mechanical stress.ST2 is an interleukin-1 receptor family member and exists in bothmembrane-bound isoform and a soluble isoform (sST2). In the context ofthe present invention, the amount of soluble ST2 shall be determined(see Dieplinger et al. (Clinical Biochemistry, 43, 2010: 1169 to 1170).ST2 also known as Interleukin 1 receptor-like 1 or IL1RL1, is encoded inhumans by the IL1RL1 gene. The sequence of the human ST2 polypeptide iswell known in the art, and e.g. accessible via GenBank, see NP_003847.2GI:27894328. Soluble ST2 (sST2) is believed to function as a decoyreceptor by binding IL-33 and abrogating the otherwise cardioprotectiveeffect of IL-33 signaling through the cell membrane-bound form of ST2.

CRP, herein also referred to as C-reactive protein, is an acute phaseprotein that was discovered more than 75 years ago to be a blood proteinthat binds to the C-polysaccharide of pneumococci. CRP is known as areactive inflammatory marker and is produced by a distal organ (i.e. theliver) in response or reaction to chemokines or interleukins originatingfrom the primary lesion site. CRP consists of five single subunits,which are non covalently linked and assembled as a cyclic pentamer witha molecular weight of approximately 110-140 kDa. Preferably, CRP as usedherein relates to human CRP. The sequence of human CRP is well known anddisclosed, e.g., by Woo et al. (J. Biol. Chem. 1985. 260 (24),13384-13388). The level of CRP is usually low in normal individuals butcan rise 100- to 200-fold or higher due to inflammation, infection orinjury (Yeh (2004) Circulation. 2004; 109:11-11-11-14). It is known thatCRP is an independent factor for the prediction of a cardiovascularrisk. Particularly, it has been shown that CRP is suitable as apredictor for myocardial infarction, stroke, peripheral arterial diseaseand sudden cardiac death. Moreover, elevated CRP amounts may alsopredict recurrent ischemia and death in patients with acute coronarysyndrome (ACS) and those undergoing coronary intervention. Determinationof CRP is recommended by expert panels (e.g. by the American HeartAssociation) in patients with a risk of coronary heart disease (see alsoPearson et al. (2003) Markers of Inflammation and CardiovascularDisease. Circulation, 107: 499-511). The term CRP also relates tovariants thereof.

Preferably, the amount of CRP in a sample of a patient is determined byusing CRP assays with a high sensitivity. The CRP determined by suchassays is frequently also referred to as high sensitivity CRP (hsCRP).hsCRP assays are, e.g., used to predict the risk of heart disease.Suitable hsCRP assays are known in the art. A particularly preferredhsCRP assay in the context of the present invention is the Roche/HitachiCRP (Latex) HS test with a detection limit of 0.1 mg/l.

Interleukin-6 (abbreviated as IL-6) is an interleukin is secreted by Tcells and macrophages to stimulate immune response, e.g. duringinfection and after trauma, especially burns or other tissue damageleading to inflammation. It acts as both a pro-inflammatory andanti-inflammatory cytokine. In humans, it is encoded by the IL6 gene.The sequence of human IL-6 can be assessed via GenBank (see NM_000600.3for the polynucleotide sequence, and NP_000591.1 for the amino acidsequence). IL-6 signals through a cell-surface type I cytokine receptorcomplex consisting of the ligand-binding IL-6Rα chain (CD126), and thesignal-transducing component gp130 (also called CD130). CD130 is thecommon signal transducer for several cytokines including leukemiainhibitory factor (LIF), ciliary neurotropic factor, oncostatin M, IL-11and cardiotrophin-1, and is almost ubiquitously expressed in mosttissues. In contrast, the expression of CD126 is restricted to certaintissues. As IL-6 interacts with its receptor, it triggers the gp130 andIL-6R proteins to form a complex, thus activating the receptor. Thesecomplexes bring together the intracellular regions of gp130 to initiatea signal transduction cascade through certain transcription factors,Janus kinases (JAKs) and Signal Transducers and Activators ofTranscription.

The marker Cystatin C is well known in the art. Cystatin C is encoded bythe CST3 gene and is produced by all nucleated cells at a constant rateand the production rate in humans is remarkably constant over the entirelifetime. Elimination from the circulation is almost entirely viaglomerular filtration. For this reason the serum concentration ofcystatin C is independent from muscle mass and gender in the age range 1to 50 years. Therefore cystatin C in plasma and serum has been proposedas a more sensitive marker for GFR. The sequence of the human Cystatin Cpolypeptide can be assessed via Genbank (see e.g. accession numberNP_000090.1). The biomarker can be determined by particle enhancedimmunoturbidimetric assay. Human cystatin C agglutinates with latexparticles coated with anti-cystatin C antibodies. The aggregate isdetermined turbidimetrically.

The marker Prealbumin is well known by the skilled person. It is atryptophan-rich protein which is synthesized in hepatocytes and has amolar mass of 55000 daltons. At a pH of 8.6, an electrophoretic bandappears prior to albumin in a relative amount of <2.5% due to itsgreater rate of diffusion to the anode. Its function is to bind andtransport low molecular weight retinol-binding proteins (molar mass ofless than 21000 daltons), preventing their glomerular filtration. 30-50%of circulating prealbumin is complexed by retinol-binding protein.Furthermore, it binds and transports thyroxine (T4), nevertheless itsaffinity to this hormone is less than that of thyroxine-bindingglobulin. The sequence of the human Prealbumin polypeptide can beassessed via Genbank (see e.g. accession number NP_000362.1). Variousmethods are available for the determination of prealbumin, such asradial immunodiffusion (RID), nephelometry and turbidimetry.

The marker “creatinine” is well known in the art. In muscle metabolism,creatinine is synthesized endogenously from creatine and creatinephosphate. Under conditions of normal renal function, creatinine isexcreted by glomerular filtration. Creatinine determinations areperformed for the diagnosis and monitoring of acute and chronic renaldisease as well as for the monitoring of renal dialysis. Creatinineconcentrations in urine can be used as reference values for theexcretion of certain analytes (albumin, α-amylase). Creatinine can bedetermined as described by Popper et al., (Popper H et al. Biochem Z1937; 291:354), Seelig and Wüst (Seelig H P, Wüst H. Ärztl Labor 1969;15:34) or Bartels (Bartels H et al. Clin Chim Acta 1972; 37:193). Forexample, sodium hydroxide and picric acid are added to the sample tostart the formation of creatinine-picric acid complex. In alkalinesolution, creatinine forms a yellow-orange complex with picrate. Thecolor intensity is directly proportional to the creatinine concentrationand can be measured photometrically.

Uric acid is the final product of purine metabolism in a subjectorganism. The IUPAC name is 7,9-dihydro-3H-purine-2,6,8-trione. Thecompound is frequently also referred to as urate, Lithic acid,2,6,8-trioxypurine, 2,6,8-trihydroxypurine, 2,6,8-Trioxopurine,1H-Purine-2,6,8-triol (compound formula C₅H₄N₄O₃, PubChem CID 1175, CASnumber 69-93-2).

Uric acid measurements are used in the diagnosis and treatment ofnumerous renal and metabolic disorders, including renal failure, gout,leukemia, psoriasis, starvation or other wasting conditions, and ofpatients receiving cytotoxic drugs. The oxidation of uric acid providesthe basis for two approaches to the quantitative determination of thispurine metabolite. One approach is the reduction of phosphotungstic acidin an alkaline solution to tungsten blue, which is measuredphotometrically. A second approach, described by Praetorius and Poulson,utilizes the enzyme uricase to oxidize uric acid; this method eliminatesthe interferences intrinsic to chemical oxidation (Praetorius E, PoulsenH. Enzymatic Determination of Uric Acid with Detailed Directions.Scandinav J Clin Lab Investigation 1953; 3:273-280). Uricase can beemployed in methods that involve the UV measurement of the consumptionof uric acid or in combination with other enzymes to provide acolorimetric assay. Another method is the colorimetric method developedby Town et al. (Town M H, Gehm S, Hammer B, Ziegenhorn J. J Clin ChemClin Biochem 1985; 23:591). The sample is initially incubated with areagent mixture containing ascorbate oxidase and a clearing system. Inthis test system it is important that any ascorbic acid present in thesample is eliminated in the preliminary reaction; this precludes anyascorbic acid interference with the subsequent POD indicator reaction.Upon addition of the starter reagent, oxidation of uric acid by uricasebegins.

In the context of the present invention, uric acid can be determined byany method deemed appropriate. Preferably, the biomarker is determinedby the aforementioned methods. More preferably, uric acid is determinedby applying a slight modification of the colorimetric method describedabove. In this reaction, the peroxide reacts in the presence ofperoxidase (POD), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline(TOOS), and 4-aminophenazone to form a quinone-diimine dye. Theintensity of the red color formed is proportional to the uric acidconcentration and is determined photometrically.

Urea is the major end product of protein nitrogen metabolism. It has thechemical formula CO(NH₂)₂ and is synthesized by the urea cycle in theliver from ammonia which is produced by amino acid deamination. Urea isexcreted mostly by the kidneys but minimal amounts are also excreted insweat and degraded in the intestines by bacterial action. Determinationof blood urea nitrogen is the most widely used screening test for renalfunction. Urea can be measured by an in vitro test for the quantitativedetermination of urea/urea nitrogen in human serum, plasma and urine onRoche/Hitachi cobas c systems. The test can be carried out automaticallyusing different analysers including cobas c 311 and cobas c 501/502. Theassay is a kinetic assay with urease and glutamate dehydrogenase. Ureais hydrolyzed by urease to form ammonium and carbonate. In the secondreaction 2-oxoglutarate reacts with ammonium in the presence ofglutamate dehydrogenase (GLDH) and the coenzyme NADH to produceL-glutamate. In this reaction 2 moles of NADH are oxidized to NAD⁺ foreach mole of urea hydrolyzed. The rate of decrease in the NADHconcentration is directly proportional to the urea concentration in thespecimen and is measured photometrically.

The marker Glucose is well known in the art as well. As used herein, themarker preferably refers to D-Glucose. The level of the marker can bedetermined by well known methods. For example, the marker can bephosphorylated to D-glucose-6-phosphate in the presence of the enzymehexokinase (HK) and adenosine-5′-triphosphate (ATP) with thesimultaneous formation of adenosine-5′-diphosphate (ATP). In the presentof the enzyme glucose-6-phosphate dehydrogenase, D-glucose-6-phosphateis oxidized to by NADP to D-gluconate phosphate with the formation ofreduced nicotinamide-adenine dinucleotide phosphate (NADPH). The amountof NADPH formed in this reaction is stoichiometric to the amount ofD-glucose. NADPH can be measured by means of light absorbance.

The marker Sodium is well known in the art. Sodium is the majorextracellular cation and functions to maintain fluid distribution andosmotic pressure. Some causes of decreased levels of sodium includeprolonged vomiting or diarrhea, diminished reabsorption in the kidneyand excessive fluid retention. Common causes of increased sodium includeexcessive fluid loss, high salt intake and increased kidneyreabsorption. The level of the marker can be determined by applying anIon Selective Electrode (ISE) which makes use of the unique propertiesof certain membrane materials to develop an electrical potential(electromotive force, EMF) for the measurements of ions in solution. Theelectrode has a selective membrane in contact with both the testsolution and an internal filling solution. The internal filling solutioncontains the test ion at a fixed concentration. Because of theparticular nature of the membrane, the test ions will closely associatewith the membrane on each side. The membrane EMF is determined by thedifference in concentration of the test ion in the test solution and theinternal filling solution. The EMF develops according to a Nernstequation for a specific ion in solution.

The marker Hemoglobin (Hb) is well known in the art. Hemoglobincomprises four protein subunits, each containing a heme moiety, and isthe red-pigmented protein located in the erythrocytes. Its main functionis to transport oxygen and carbon dioxide in blood. Each Hb molecule isable to bind four oxygen molecules. Hb consists of a variety ofsubfractions and derivatives. The term “hemoglobin” as used herein,preferably, refers to total hemoglobin. The level of Hemoglobin can bemeasured by well known methods, e.g. by oxidation of hemoglobin tomethemoglobin by potassium hexacyanoferrate (III) (Fe²⁺→Fe³⁺). Thehemoglobin level is proportional to the color intensity and, e.g., canbe measured at a wavelength of 567 nm and 37° C. The level of hemoglobincan be also measured by contacting the sample with an antibody whichspecifically binds to hemoglobin.

The marker HbA1c (glycated hemoglobin, Glycohemoglobin) is well known inthe art as well. HbA1c is one of the glycated hemoglobins, a subfractionformed by the attachment of various sugars to the Hb molecule. HbA1c isformed in two steps by the nonenzymatic reaction of glucose with theN-terminal amino group of the β-chain of normal adult Hb (HbA). Thefirst step is reversible and yields labile HbA1c. This is rearranged toform stable HbA1c in a second reaction step. In the erythrocytes, therelative amount of HbA converted to stable HbA1c increases with theaverage concentration of glucose in the blood. The conversion to stableHbA1c is limited by the erythrocyte's life span of approximately 100 to120 days. The level of Hemoglobin can be measured by well known methods.Preferably, the measurement of the level of HbA1c encompasses themeasurement of the level of all hemoglobin variants which are glycatedat the β-chain N-terminus of HbA (adult hemoglobin). In an embodimentthe level of this marker is measured by contacting the sample with anantibody which specifically binds to this marker. In this case,Clycohemoglobin (HbA1c) in the sample reacts with anti-HbA1c antibody toform soluble antigen-antibody complexes

Hematocrit (Ht or HCT) also known as packed cell volume (PCV) orerythrocyte volume fraction (EVF), is the volume percentage (%) of redblood cells in blood. As used, the term “hematocrit”, preferably, refersto the percentage of packed red blood cells in a volume of whole blood.Hematocrit can be determined by centrifuging heparinized blood in acapillary tube (also known as a microhematocrit tube) at 10,000 RPM forfive minutes. This separates the blood into layers. The volume of packedred blood cells divided by the total volume of the blood sample givesthe PCV. Because a tube is used, this can be calculated by measuring thelengths of the layers. With modern lab equipment, the hematocrit iscalculated by an automated analyzer and not directly measured. It isdetermined by multiplying the red cell count by the mean cell volume.The hematocrit is slightly more accurate as the PCV includes smallamounts of blood plasma trapped between the red cells. An estimatedhematocrit as a percentage may be derived by tripling the hemoglobinconcentration in g/dL and dropping the units.

The term “QRS duration” is well known in the art. The QRS duration is astandard measure in medicine and describes the duration of the QRS groupon the surface electrocardiogram (ECG) that is indication the durationof electrical excitation of the ventricles. Preferably, the QRS durationis measured by an ECG device.

The biomarkers as referred to herein can be detected using methodsgenerally known in the art. Methods of detection generally encompassmethods to quantify the level of a biomarker in the sample (quantitativemethod). It is generally known to the skilled artisan which of thefollowing methods are suitable for qualitative and/or for quantitativedetection of a biomarker. Samples can be conveniently assayed for, e.g.,proteins using Westerns and immunoassays, like ELISAs, RIAs,fluorescence-based immunoassays, which are commercially available.Further suitable methods to detect biomarker include measuring aphysical or chemical property specific for the peptide or polypeptidesuch as its precise molecular mass or NMR spectrum. Said methodscomprise, e.g., biosensors, optical devices coupled to immunoassays,biochips, analytical devices such as mass-spectrometers, NMR-analyzers,or chromatography devices. Further, methods include microplateELISA-based methods, fully-automated or robotic immunoassays (availablefor example on Elecsys™ analyzers), CBA (an enzymatic Cobalt BindingAssay, available for example on Roche-Hitachi™ analyzers), and latexagglutination assays (available for example on Roche-Hitachi™analyzers).

For the detection of biomarker proteins as referred to herein a widerange of immunoassay techniques using such an assay format areavailable, see, e.g., U.S. Pat. Nos. 4,016,043, 4,424,279, and4,018,653. These include both single-site and two-site or “sandwich”assays of the non-competitive types, as well as in the traditionalcompetitive binding assays. These assays also include direct binding ofa labeled antibody to a target biomarker.

Sandwich assays are among the most useful and commonly usedimmunoassays.

Methods for measuring electrochemiluminescent phenomena are well-known.Such methods make use of the ability of special metal complexes toachieve, by means of oxidation, an excited state from which they decayto ground state, emitting electrochemiluminescence. For review seeRichter, M. M., Chem. Rev. 104 (2004) 3003-3036.

Biomarkers can also be detected by generally known methods includingmagnetic resonance spectroscopy (NMR spectroscopy), Gaschromatography-mass spectrometry (GC-MS), Liquid chromatography-massspectrometry (LC-MS), High and ultra-HPLC HPLC such as reverse phaseHPLC, for example, ion-pairing HPLC with dual UV-wavelength detection,capillary electrophoresis with laser-induced fluorescence detection,anion exchange chromatography and fluorescent detection, thin layerchromatography.

Preferably, measuring the level of a biomarker as referred to hereincomprises the steps of (a) contacting a cell capable of eliciting acellular response the intensity of which is indicative of the level ofthe peptide or polypeptide with the said peptide or polypeptide for anadequate period of time, (b) measuring the cellular response. Formeasuring cellular responses, the sample or processed sample is,preferably, added to a cell culture and an internal or external cellularresponse is measured. The cellular response may include the measurableexpression of a reporter gene or the secretion of a substance, e.g. apeptide, polypeptide, or a small molecule. The expression or substanceshall generate an intensity signal which correlates to the level of thepeptide or polypeptide.

Also preferably, measuring the level of a peptide or polypeptidecomprises the step of measuring a specific intensity signal obtainablefrom the peptide or polypeptide in the sample. As described above, sucha signal may be the signal intensity observed at an m/z variablespecific for the peptide or polypeptide observed in mass spectra or aNMR spectrum specific for the peptide or polypeptide.

Measuring the level of a peptide or polypeptide may, preferably,comprises the steps of (a) contacting the peptide with a specificbinding agent, (b) (optionally) removing non-bound binding agent, (c)measuring the level of bound binding agent, i.e. the complex of thebinding agent formed in step (a). According to a preferred embodiment,said steps of contacting, removing and measuring may be performed by ananalyzer unit of the system disclosed herein. According to someembodiments, said steps may be performed by a single analyzer unit ofsaid system or by more than one analyzer unit in operable communicationwith each other. For example, according to a specific embodiment, saidsystem disclosed herein may include a first analyzer unit for performingsaid steps of contacting and removing and a second analyzer unit,operably connected to said first analyzer unit by a transport unit (forexample, a robotic arm), which performs said step of measuring.

The bound binding agent, i.e. the binding agent or the bindingagent/peptide complex, will generate an intensity signal. Bindingaccording to the present invention includes both covalent andnon-covalent binding. A binding agent according to the present inventioncan be any compound, e.g., a peptide, polypeptide, nucleic acid, orsmall molecule, binding to the peptide or polypeptide described herein.Preferred binding agents include antibodies, nucleic acids, peptides orpolypeptides such as receptors or binding partners for the peptide orpolypeptide and fragments thereof comprising the binding domains for thepeptides, and aptamers, e.g. nucleic acid or peptide aptamers. Methodsto prepare such binding agents are well-known in the art. For example,identification and production of suitable antibodies or aptamers is alsooffered by commercial suppliers. The person skilled in the art isfamiliar with methods to develop derivatives of such binding agents withhigher affinity or specificity. For example, random mutations can beintroduced into the nucleic acids, peptides or polypeptides. Thesederivatives can then be tested for binding according to screeningprocedures known in the art, e.g. phage display. Antibodies as referredto herein include both polyclonal and monoclonal antibodies, as well asfragments thereof, such as Fv, Fab and F(ab)2 fragments that are capableof binding antigen or hapten. The present invention also includes singlechain antibodies and humanized hybrid antibodies wherein amino acidsequences of a non-human donor antibody exhibiting a desiredantigen-specificity are combined with sequences of a human acceptorantibody. The donor sequences will usually include at least theantigen-binding amino acid residues of the donor but may comprise otherstructurally and/or functionally relevant amino acid residues of thedonor antibody as well. Such hybrids can be prepared by several methodswell known in the art. Preferably, the binding agent or agent bindsspecifically to the pep-tide or polypeptide. Specific binding accordingto the present invention means that the ligand or agent should not bindsubstantially to (“cross-react” with) another peptide, polypeptide orsubstance present in the sample to be analyzed. Preferably, thespecifically bound peptide or polypeptide should be bound with at least3 times higher, more preferably at least 10 times higher and even morepreferably at least 50 times higher affinity than any other relevantpeptide or polypeptide. Non-specific binding may be tolerable, if it canstill be distinguished and measured unequivocally, e.g. according to itssize on a Western Blot, or by its relatively higher abundance in thesample.

Binding of the binding agent can be measured by any method known in theart. Preferably, said method is semi-quantitative or quantitative.Further suitable techniques for the determination of a polypeptide orpeptide are described in the follow-ing.

Binding of a binding agent may be measured directly, e.g. by NMR orsurface plasmon resonance. Measurement of the binding of a bindingagent, according to preferred embodiments, is performed by an analyzerunit of a system disclosed herein. Thereafter, a level of the measuredbinding may be calculated by a computing device of a system disclosedherein. If the binding agent also serves as a substrate of an enzymaticactivity of the pep-tide or polypeptide of interest, an enzymaticreaction product may be measured (e.g. the level of a protease can bemeasured by measuring the level of cleaved substrate, e.g. on a WesternBlot). Alternatively, the binding agent may exhibit enzymatic propertiesitself and the “binding agent/peptide or polypeptide” complex or thebinding agent which was bound by the peptide or polypeptide,respectively, may be contacted with a suitable substrate allowingdetection by the generation of an intensity signal. For measurement ofenzymatic reaction products, preferably the level of substrate issaturating. The substrate may also be labeled with a detectable labelprior to the reaction. Preferably, the sample is contacted with thesubstrate for an adequate period of time. An adequate period of timerefers to the time necessary for a detectable, preferably measurable,level of product to be produced. Instead of measuring the level ofproduct, the time necessary for appearance of a given (e.g. detectable)level of product can be measured. Third, the binding agent may becoupled covalently or non-covalently to a label allowing detection andmeasurement of the binding agent. Labeling may be done by direct orindirect methods. Direct labeling involves coupling of the labeldirectly (covalently or non-covalently) to the binding agent. Indirectlabeling involves binding (covalently or non-covalently) of a secondarybinding agent to the first binding agent. The secondary binding agentshould specifically bind to the first binding agent. Said secondarybinding agent may be coupled with a suitable label and/or be the target(receptor) of tertiary binding agent binding to the secondary bindingagent. The use of secondary, tertiary or even higher order bindingagents is often used to increase the signal. Suitable secondary andhigher order binding agents may include antibodies, secondaryantibodies, and the well-known streptavidin-biotin system (VectorLa-boratories, Inc.). The binding agent or substrate may also be“tagged” with one or more tags as known in the art. Such tags may thenbe targets for higher order binding agents. Suitable tags includebiotin, digoxygenin, His-Tag, Glutathion-S-Transferase, FLAG, GFP,myc-tag, influenza A virus haemagglutinin (HA), maltose binding protein,and the like. In the case of a peptide or polypeptide, the tag ispreferably at the N-terminus and/or C-terminus. Suitable labels are anylabels detectable by an appropriate detection method. Typical labelsinclude gold particles, latex beads, acridan ester, luminol, ruthenium,enzymatically active labels, radioactive labels, magnetic labels (“e.g.magnetic beads”, including paramagnetic and superparamagnetic labels),and fluo-rescent labels. Enzymatically active labels include e.g.horseradish peroxidase, alkaline phosphatase, beta-Galactosidase,Luciferase, and derivatives thereof. Suitable substrates for detectioninclude di-amino-benzidine (DAB), 3,3′-5,5′-tetramethylbenzidine,NBT-BCIP (4-nitro blue tetrazolium chloride and5-bromo-4-chloro-3-indolyl-phosphate, avail-able as ready-made stocksolution from Roche Diagnostics), CDP-Star™ (Amersham Bio-sciences),ECF™ (Amersham Biosciences). A suitable enzyme-substrate combination mayresult in a colored reaction product, fluorescence or chemoluminescence,which can be measured according to methods known in the art (e.g. usinga light-sensitive film or a suit-able camera system). As for measuringthe enzymatic reaction, the criteria given above apply analogously.Typical fluorescent labels include fluorescent proteins (such as GFP andits derivatives), Cy3, Cy5, Texas Red, Fluorescein, and the Alexa dyes(e.g. Alexa 568). Further fluorescent labels are available e.g. fromMolecular Probes (Oregon). Also the use of quantum dots as fluorescentlabels is contemplated. A radioactive label can be detected by anymethod known and appropriate, e.g. a light-sensitive film or a phosphorimager.

The level of a peptide or polypeptide may be, also preferably,determined as follows: (a) contacting a solid support comprising abinding agent for the peptide or polypeptide as specified above with asample comprising the peptide or polypeptide and (b) measuring the levelpeptide or polypeptide which is bound to the support. The binding agent,preferably chosen from the group consisting of nucleic acids, peptides,polypeptides, antibodies and aptamers, is preferably present on a solidsupport in immobilized form. Materials for manufacturing solid supportsare well known in the art and include, inter alia, commerciallyavailable column materials, polystyrene beads, latex beads, magneticbeads, colloid metal particles, glass and/or silicon chips and surfaces,nitrocellulose strips, membranes, sheets, duracytes, wells and walls ofreaction trays, plastic tubes etc. The binding agent or agent may bebound to many different carriers. Examples of well-known carriersinclude glass, polystyrene, polyvinyl chloride, polypropylene,polyethylene, polycarbonate, dextran, nylon, amyloses, natural andmodified celluloses, polyacrylamides, agaroses, and magnetite. Thenature of the carrier can be either soluble or insoluble for thepurposes of the invention. Suitable methods for fixing/immobilizing saidbinding agent are well known and include, but are not limited to ionic,hydrophobic, covalent interactions and the like. It is also contemplatedto use “suspension arrays” as arrays according to the present invention(Nolan 2002, Trends Biotechnol. 20(1):9-12). In such suspension arrays,the carrier, e.g. a mi-crobead or microsphere, is present in suspension.The array consists of different microbeads or microspheres, possiblylabeled, carrying different binding agents. Methods of producing sucharrays, for example based on solid-phase chemistry and photo-labileprotective groups, are generally known (U.S. Pat. No. 5,744,305).

In an embodiment of the method of the present invention, the levels ofthe biomarkers as referred to herein are measured by using the assaysdescribed in the Examples section.

In another embodiment of the method of the present invention, themeasurement in step a) may be carried out by an analyzer unit, inparticular by an analyzer unit as defined elsewhere herein.

The term “binding agent” refers to a molecule that comprises a bindingmoiety which specifically binds the corresponding to the respectivebiomarker. Examples of “binding agent” are a aptamer, antibody, antibodyfragment, peptide, peptide nucleic acid (PNA) or chemical compound.

The term “specific binding” or “specifically bind” refers to a bindingreaction wherein binding pair molecules exhibit a binding to each otherunder conditions where they do not significantly bind to othermolecules. The term “specific binding” or “specifically binds”, whenreferring to a protein or peptide as biomarker, refers to a bindingreaction wherein a binding agent binds to the corresponding biomarkerwith an affinity of at least 10-7 M. The term “specific binding” or“specifically binds” preferably refers to an affinity of at least 10-8 Mor even more preferred of at least 10-9 M for its target molecule. Theterm “specific” or “specifically” is used to indicate that othermolecules present in the sample do not significantly bind to the bindingagent specific for the target molecule. Preferably, the level of bindingto a molecule other than the target molecule results in a bindingaffinity which is only 10% or less, more preferably only 5% or less ofthe affinity to the target molecule.

Examples of “binding agents” are a nucleic acid probe, nucleic acidprimer, DNA molecule, RNA molecule, aptamer, antibody, antibodyfragment, peptide, peptide nucleic acid (PNA) or chemical compound. Apreferred binding agent is an antibody which specifically binds to thebiomarker to be measured. The term “antibody” herein is used in thebroadest sense and encompasses various antibody structures, includingbut not limited to monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies), and antibodyfragments so long as they exhibit the desired antigen-binding activity.Preferably, the antibody is a polyclonal antibody. More preferably, theantibody is a monoclonal antibody.

Another binding agent that can be applied, in an aspect, may be anaptamere which specifically binds to the at least one marker in thesample. The term “specific binding” or “specifically binds”, whenreferring to a nucleic acid aptamer as a binding agent, refers to abinding reaction wherein a nucleic acid aptamer binds to thecorresponding target molecule with an affinity in the low nM to pMrange.

In yet an aspect the, sample is removed from the complex formed betweenthe binding agent and the at least one marker prior to the measurementof the level of formed complex. Accordingly, in an aspect, the bindingagent may be immobilized on a solid support. In yet an aspect, thesample can be removed from the formed complex on the solid support byapplying a washing solution. The formed complex shall be proportional tothe level of the at least one marker present in the sample. It will beunderstood that the specificity and/or sensitivity of the binding agentto be applied defines the degree of proportion of at least one markercomprised in the sample which is capable of being specifically bound.Further details on how the determination can be carried out are alsofound elsewhere herein. The level of formed complex shall be transformedinto a level of at least one marker reflecting the level indeed presentin the sample. Such a level, in an aspect, may be essentially the amountpresent in the sample or may be, in another aspect, an amount which is acertain proportion thereof due to the relationship between the formedcomplex and the amount present in the original sample.

The term “level” as used herein encompasses the absolute amount of abiomarker as referred to herein, the relative amount or concentration ofthe said biomarker as well as any value or parameter which correlatesthereto or can be derived therefrom. Such values or parameters compriseintensity signal values from all specific physical or chemicalproperties obtained from the said peptides by direct measurements, e.g.,intensity values in mass spectra or NMR spectra. Moreover, encompassedare all values or parameters which are obtained by indirect measurementsspecified elsewhere in this description, e.g., response amountsdetermined from biological read out systems in response to the peptidesor intensity signals obtained from specifically bound ligands. It is tobe understood that values correlating to the aforementioned amounts orparameters can also be obtained by all standard mathematical operations.

The term “comparing” as used herein refers to comparing the level of thebiomarkers in the sample from the individual or patient with thereference level of the biomarkers specified elsewhere in thisdescription. It is to be understood that comparing as used hereinusually refers to a comparison of corresponding parameters or values,e.g., an absolute amount is compared to an absolute reference amountwhile a concentration is compared to a reference concentration or anintensity signal obtained from the biomarker in a sample is compared tothe same type of intensity signal obtained from a reference sample. Thecomparison may be carried out manually or computer assisted. Thus, thecomparison may be carried out by a computing device (e.g., of a systemdisclosed herein). The value of the measured or detected level of thebiomarker in the sample from the individual or patient and the referencelevel can be, e.g., compared to each other and the said comparison canbe automatically carried out by a computer program executing analgorithm for the comparison. The computer program carrying out the saidevaluation will provide the desired assessment in a suitable outputformat. For a computer assisted comparison, the value of the determinedamount may be compared to values corresponding to suitable referenceswhich are stored in a database by a computer program. The computerprogram may further evaluate the result of the comparison, i.e.automatically provide the desired assessment in a suitable outputformat. For a computer assisted comparison, the value of the determinedamount may be compared to values corresponding to suitable referenceswhich are stored in a database by a computer program. The computerprogram may further evaluate the result of the comparison, i.e.automatically provides the desired assessment in a suitable outputformat.

In certain embodiments, the term “reference level” herein refers to apredetermined value for the respective biomarker. In this context“level” encompasses the absolute amount, the relative amount orconcentration as well as any value or parameter which correlates theretoor can be derived therefrom. Preferably, the reference level is a levelwhich allows for allocating the patient into a group of patients beingeligible to intensification of heart failure therapy, or into a group ofpatients not being eligible to intensification of heart failure therapy.Thus, the reference level shall allow for differentiating between apatient who is eligible to intensification of heart failure therapy anda patient who is not eligible to intensification of heart failuretherapy.

As the skilled artisan will appreciate the reference level ispredetermined and set to meet routine requirements in terms of e.g.specificity and/or sensitivity. These requirements can vary, e.g. fromregulatory body to regulatory body. It may for example be that assaysensitivity or specificity, respectively, has to be set to certainlimits, e.g. 80%, 90%, 95% or 98%, respectively. These requirements mayalso be defined in terms of positive or negative predictive values.Nonetheless, based on the teaching given in the present invention itwill always be possible for a skilled artisan to arrive at the referencelevel meeting those requirements. In one embodiment the reference levelis determined in a reference sample or samples from a patient (or groupof patients) having heart failure and being eligible to intensificationof heart failure therapy or in a reference sample or samples from apatient (or group of patients) having heart failure and not beingeligible to intensification of heart failure therapy. The referencelevel in one embodiment has been predetermined in reference samples fromthe disease entity to which the patient belongs. In certain embodimentsthe reference level can e.g. be set to any percentage between 25% and75% of the overall distribution of the values in a disease entityinvestigated. In other embodiments the reference level can e.g. be setto the median, tertiles or quartiles as deter-mined from the overalldistribution of the values in reference samples from a disease entityinvestigated. In one embodiment the reference level is set to the medianvalue as determined from the overall distribution of the values in adisease entity investigated. The reference level may vary depending onvarious physiological parameters such as age, gender or subpopulation,as well as on the means used for the determination of the biomarkersreferred to herein. In one embodiment, the reference sample is fromessentially the same type of cells, tissue, organ or body fluid sourceas the sample from the individual or patient subjected to the method ofthe invention, e.g. if according to the invention blood is used as asample to determine the level of biomarkers in the individual, thereference level is also determined in blood or a part thereof.

In certain embodiments, the term “larger than the reference level” or“above the reference level” refers to a level of the biomarker in thesample from the individual or patient above the reference level or to anoverall increase of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%,85%, 90%, 95%, 100% or greater, determined by the methods describedherein, as compared to the reference level. In certain embodiments, theterm increase refers to the increase in biomarker level in the samplefrom the individual or patient wherein, the increase is at least about1.5-, 1.75-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-,40-, 50-, 60-, 70-, 75-, 80-, 90-, or 100-fold higher as compared to thereference level, e.g. predetermined from a reference sample.

In certain embodiments, the term “lower than the reference level” or“below” herein refers to a level of the biomarker in the sample from theindividual or patient below the reference level or to an overallreduction of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or greater, determined by the methods describedherein, as compared to the reference level. In certain embodiments, theterm decrease in biomarker level in the sample from the individual orpatient wherein the decreased level is at most about 0.9-, 0.8-, 0.7-,0.6-, 0.5-, 0.4-, 0.3-, 0.2-, 0.1-, 0.05-, or 0.01-fold of the referencelevel, e.g. predetermined from a reference sample, or lower.

The following applies as diagnostic algorithm if the at least one markeris selected from the group consisting of creatinine, urea, glucose,HbA1c (glycated hemoglobin), CRP (C-reactive protein, in particular highsensitive CRP), Cystatin C, IL-6 (Interleukin 6), Prealbumin, sFlt-1(soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (GrowthDifferentiation Factor 15), Galectin-3 (Gal-3), Endostatin, Mimecan,sST2 (soluble ST2), and Osteopontin: Preferably, a level (levels) of theat least one marker in the sample from the patient which is above thereference level (reference levels) for said marker (markers) indicatesthat the patient is eligible to intensification of heart failuretherapy, and/or a level (levels) of the at least one marker in thesample from the patient which is below the reference level (referencelevels) for said marker (markers) indicates that the patient is noteligible to intensification of heart failure therapy.

The following applies as diagnostic algorithm if the at least one markeris selected from the group consisting of sodium, hemoglobin, hematocrit,and IGFBP-7:

Preferably, a level (levels) of the at least one marker in the samplefrom the patient which is below the reference level (reference levels)for said marker (markers) indicates that the patient is eligible tointensification of heart failure therapy, and/or a level (levels) of theat least one marker in the sample from the patient which is above thereference level (reference levels) for said marker (markers) indicatesthat the patient is not eligible to intensification of heart failuretherapy.

The following table A provides preferred ranges for reference levels(third column) for the various markers as well as preferred specificreference levels (fourth column). The person skilled in the art candetermine further reference levels without further ado.

TABLE A Parameter reference level within Marker Units the range of fromreference level Creatinine mg/dL about 1.2 to 1.8 about 1.5 BUN (urea)mmol/L about 10 to 12 about 11.1 Glucose mmol/L about 10 to 13 about11.6 HbA1c % about 0.05 to 0.07 about 0.06 hsCRP mg/mL about 9 to 13about 10.4 Cystatin C mg/L about 1.8 to 2.0 about 1.9 IL-6 pg/mL about 8to 10 about 9.1 Prealbumin g/L about 0.14 to 0.18 about 0.16 sFLt-1pg/mL about 85 to 100 about 87 Uric Acid mg/dL about 9 to 10 about 9.1GFD-15 pg/mL about 2500 to 5000 about 3210 sST2 ng/mL about 38 to 47about 41.5 Galectin-3 ng/mL about 24 to 30 about 25 Endostatin ng/mLabout 230 to 277 about 243 Mimecan ng/mL about 44 to 50 about 45.2IGFBP-7 ng/mL about 70 to 77 about 71.4 Osteopontin ng/mL about 110 to120 about 113.5 Hemoglobin g/dL about 7.5 to 8.5 about 8.04 Hematocrit %about 0.37 to 0.43 about 0.40 QRS duration ms about 140 to 180 about 160Sodium mmol/L about 138 to 142 about 141

As regards to the QRS duration, the reference may be in the range ofabout 140 to about 180 ms. In an embodiment, the reference is about 160ms.

In the context of the present invention, it is envisaged to measure thelevel of a single marker, or of a combination of markers. Thus, it isenvisaged to measure the level of two, three, four or of even moremarkers. Preferred combinations are as follows:

For example, the following marker combinations are envisaged:

-   -   creatinine and sodium    -   hemoglobin and QRS duration    -   urea and HbA1c    -   hematocrit and creatinine

In accordance with the present invention, the level of a certain markermay be measured in order to identify a patient who is eligible tointensification of a certain heart failure therapy, in particular theintensified treatment with a certain medicament. For example, a markermay be used in order to assess whether the treatment with the medicamentshall be intensified or not (e.g. whether the dosage of the administeredmedicament shall be increased or not). For example, if the marker to bemeasured is creatinine, the heart failure therapy to be intensified,preferably is treatment with a beta blocker.

The definitions given herein above apply mutatis mutandis to thefollowing. Also, the steps carried out in connection with the methoddescribed herein above, may be carried out in accordance with thefollowing method.

The present invention also relates to a method, in particular an invitro method, for identifying a patient who is eligible to anintensification of heart failure therapy, said method comprising thestep of

-   -   (a) measuring the level of a BNP-type peptide in a in a sample        from a patient who has heart failure and who receives BNP-type        peptide guided heart failure therapy;    -   (b) measuring the level of at least one marker selected from the        group consisting of creatinine, urea, sodium, glucose, HbA1c        (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive        protein, in particular high sensitive CRP), Cystatin C, IL-6        (Interleukin 6), Prealbumin, sFlt-1 (soluble fms-like tyrosine        kinase-1), uric acid, GDF-15 (Growth Differentiation Factor 15),        Galectin-3 (Gal-3), Endostatin, Mimecan, IGFBP7 (Insulin Growth        Factor Binding Protein 7), sST2 (soluble ST2), and Osteopontin        in a sample from the patient,    -   (c) comparing the level of the BNP-type peptide measured in (a)        to a reference level (or reference levels), and    -   (d) comparing the level (or levels) of the at least one marker        measured in (b) to a reference level (or reference levels).

By carrying out steps (c) or (d), a patient who is eligible tointensification of heart failure therapy is identified. In anembodiment, the method further comprises step (e) of identifying orselecting a patient who is eligible to an intensification of heartfailure therapy. In addition, the method may comprise step (f) ofintensifying heart failure therapy or recommending intensification ofheart failure therapy (if the patient is identified as to be eligible tointensification of heart failure therapy). Accordingly, the presentinvention also envisages also a method of intensifying heart failuretherapy, said method comprising steps (a) to (f) as set forth above.

In addition the a marker as referred to step a), or alternatively, theQRS duration may be measured or provided and compared to the a reference(as outlined elsewhere herein)

In addition to the method describe above, the method further comprisesthe step of measuring the level of a BNP-type peptide.

As used herein, the term “BNP-type peptides” comprise pre-proBNP,proBNP, NT-proBNP, and BNP. The pre-pro peptide (134 amino acids in thecase of pre-proBNP) comprises a short signal peptide, which isenzymatically cleaved off to release the pro peptide (108 amino acids inthe case of proBNP). The pro peptide is further cleaved into anN-terminal pro peptide (NT-pro peptide, 76 amino acids in case ofNT-proBNP) and the active hormone (32 amino acids in the case of BNP).Preferably, BNP-type peptides according to the present invention areNT-proBNP, BNP (brain natriuretic peptide), and variants thereof. BNP isthe active hormone and has a shorter half-life than the respectiveinactive counterpart NT-proBNP. BNP is metabolized in the blood, whereasNT-proBNP circulates in the blood as an intact molecule and as such iseliminated renally. The in-vivo half-life of NT-proBNP is 120 min longerthan that of BNP, which is 20 min (Smith 2000, J Endocrinol. 167:239-46.). Preanalytics are more robust with NT-proBNP allowing easytransportation of the sample to a central laboratory (Mueller 2004, ClinChem Lab Med 42: 942-4.). Blood samples can be stored at roomtemperature for several days or may be mailed or shipped withoutrecovery loss. In contrast, storage of BNP for 48 hours at roomtemperature or at 4° Celsius leads to a concentration loss of at least20% (Mueller loc.cit.; Wu 2004, Clin Chem 50: 867-73.). Therefore,depending on the time-course or properties of interest, eithermeasurement of the active or the inactive forms of the natriureticpeptide can be advantageous. The most preferred BNP-type peptidesaccording to the present invention are NT-proBNP or variants thereof. Asbriefly discussed above, the human NT-proBNP, as referred to inaccordance with the present invention, is a polypeptide comprising,preferably, 76 amino acids in length corresponding to the N-terminalportion of the human NT-proBNP molecule. The structure of the human BNPand NT-proBNP has been described already in detail in the prior art,e.g., WO 02/089657, WO 02/083913 or Bonow loc. cit. Preferably, humanNT-proBNP as used herein is human NT-proBNP as disclosed in EP 0 648 228B1. These prior art documents are herewith incorporated by referencewith respect to the specific sequences of NT-proBNP and variants thereofdisclosed therein. The NT-proBNP referred to in accordance with thepresent invention further encompasses allelic and other variants of saidspecific sequence for human NT-proBNP discussed above. Specifically,envisaged are variant polypeptides which are on the amino acid levelpreferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%,or 99% identical to human NT-proBNP, preferably over the entire lengthof human NT-proBNP. The degree of identity between two amino acidsequences can be determined by algorithms well known in the art.Preferably, the degree of identity is to be determined by comparing twooptimally aligned sequences over a comparison window, where the fragmentof amino acid sequence in the comparison window may comprise additionsor deletions (e.g., gaps or overhangs) as compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment. The percentage is calculated by determining the number ofpositions at which the identical amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman Add.APL. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (USA) 85:2444 (1988), by computerized implementations of these algorithms (GAP,BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.),or by visual inspection. Given that two sequences have been identifiedfor comparison, GAP and BESTFIT are preferably employed to determinetheir optimal alignment and, thus, the degree of identity. Preferably,the default values of 5.00 for gap weight and 0.30 for gap weight lengthare used. Variants referred to above may be allelic variants or anyother species specific homologs, paralogs, or orthologs. Substantiallysimilar and also envisaged are proteolytic degradation products whichare still recognized by the diagnostic means or by ligands directedagainst the respective full-length peptide. Also encompassed are variantpolypeptides having amino acid deletions, substitutions, and/oradditions compared to the amino acid sequence of human NT-proBNP as longas the said polypeptides have NT-proBNP properties. NT-proBNP propertiesas referred to herein are immunological and/or biological properties.Preferably, the NT-proBNP variants have immunological properties (i.e.epitope composition) comparable to those of human NT-proBNP. Thus, thevariants shall be recognizable by the aforementioned means or ligandsused for determination of the amount of the natriuretic peptides.Biological and/or immunological NT-proBNP properties can be detected bythe assay described in Karl et al. (Karl 1999, Scand J Clin Lab Invest230:177-181), Yeo et al. (Yeo 2003, Clinica Chimica Acta 338:107-115).Also, an assay for the determination of NT-proBNP is described byMueller T. et al., Clinica Chimica Acta 341 (2004) 41-48. In anembodiment, the NT-proBNP is carried out as described any one of theaforementioned references. Variants also include posttranslationallymodified peptides such as glycosylated peptides. Further, a variant inaccordance with the present invention is also a peptide or polypeptidewhich has been modified after collection of the sample, for example bycovalent or non-covalent attachment of a label, particularly aradioactive or fluorescent label, to the peptide.

The term “reference level” has been defined above. The reference levelfor the BNP-type peptide, preferably, shall be a level which when takenalone (i.e. not in combination with the further markers as referred inthe context of the present invention) which is indicative for a patientwho is not eligible to an intensification of heart failure therapy.Preferred reference levels for said BNP-type peptide being indicative ofintensification of heart failure therapy to be applied in the context ofthe present invention are those described in the Examples. Preferredreference levels are within a range from about 80 to 400 pg/ml, or, inparticular, from about 80 to 200 pg/ml for BNP, and within a range fromabout 450 to 2200 pg/ml, or in particular from about 800 pg/ml to 1200pg/ml for NT-proBNP. Further preferred reference levels are about 100pg/ml or 400 pg/ml for BNP, and about 1000 or 1200 pg/ml for NT-proBNP.

Preferred reference levels or ranges for references levels for themarkers creatinine, urea, sodium, glucose, HbA1c (glycated hemoglobin),hemoglobin, hematocrit, CRP (C-reactive protein, in particular highsensitive CRP), Cystatin C, IL-6 (Interleukin 6), Prealbumin, sFlt-1(soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (GrowthDifferentiation Factor 15), Galectin-3 (Gal-3), Endostatin, Mimecan,IGFBP7 (Insulin Growth Factor Binding Protein 7), sST2 (soluble ST2),and Osteopontin (OPN) are shown in Table A above.

The following applies as diagnostic algorithm if the at least one markeras measured in step (b) is selected from the group consisting ofcreatinine, urea, glucose, HbA1c (glycated hemoglobin), CRP (C-reactiveprotein, in particular high sensitive CRP), Cystatin C, IL-6(Interleukin 6), Prealbumin, sFlt-1 (soluble fms-like tyrosinekinase-1), uric acid, GDF-15 (Growth Differentiation Factor 15),Galectin-3 (Gal-3), Endostatin, Mimecan, sST2 (soluble ST2), andOsteopontin:

-   -   (a) a level of the at least one marker in the sample from the        patient which is above the reference level for said marker, and        a level of BNP-type peptide which is above the reference level        for said BNP-type peptide is indicative for a patient who is        eligible to intensification of heart failure therapy,    -   (b) a level of the at least one marker in the sample from the        patient which is above the reference level for said marker, and        a level of said BNP-type peptide which is below the reference        level for said BNP-type peptide is indicative for a patient who        is eligible to intensification of heart failure therapy,    -   (c) a level of the at least one marker in the sample from the        patient which is below the reference level for said marker, and        a level of said BNP-type peptide which is above the reference        level for said BNP-type peptide is indicative for a patient who        is eligible to intensification of heart failure therapy, and/or    -   (d) a level of the at least one marker in the sample from the        patient which is below the reference level for said marker, and        a level of said BNP-type peptide which is below the reference        level for said BNP-type peptide is indicative for a patient who        is not eligible to intensification of heart failure therapy.

Alternatively or additionally, the following applies as diagnosticalgorithm if the at least one marker as measured in step (b) is selectedfrom the group consisting of sodium, hemoglobin, hematocrit, andIGFBP-7:

-   -   (a) a level of the at least one marker in the sample from the        patient which is below the reference level for said marker, and        a level of said BNP-type peptide which is above the reference        level for said BNP-type peptide is indicative for a patient who        is eligible to intensification of heart failure therapy,    -   (b) a level of the at least one marker in the sample from the        patient which is below the reference level for said marker, and        a level of said BNP-type peptide which is below the reference        level for said BNP-type peptide is indicative for a patient who        is eligible to intensification of heart failure therapy,    -   (c) a level of the at least one marker in the sample from the        patient which is above the reference level for said marker, and        a level of said BNP-type peptide which is above the reference        level for said BNP-type peptide is indicative for a patient who        is eligible to intensification of heart failure therapy, and/or    -   (d) a level of the at least one marker in the sample from the        patient which is above the reference level for said marker, and        a level of said BNP-type peptide which is below the reference        level for said BNP-type peptide is indicative for a patient who        is not eligible to intensification of heart failure therapy.

The patient to be tested in accordance with the aforementioned methodmay display any level (in particular any blood, serum or plasma level)of a BNP-type peptide.

Moreover, the present invention relates to a method for optimizingBNP-type peptide guided heart failure therapy, said method comprisingthe steps of

-   -   (a) measuring the level of at least one marker selected from the        group consisting of creatinine, urea, sodium, glucose, HbA1c        (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive        protein, in particular high sensitive CRP), Cystatin C, IL-6        (Interleukin 6), Prealbumin, sFlt-1 (soluble fms-like tyrosine        kinase-1), uric acid, GDF-15 (Growth Differentiation Factor 15),        Galectin-3 (Gal-3), Endostatin, Mimecan, IGFBP7 (Insulin Growth        Factor Binding Protein 7), sST2 (soluble ST2), and Osteopontin        in a sample from a patient who has heart failure and who        receives BNP-type peptide guided therapy, and    -   (b) comparing the level (or levels) of the marker (or markers)        measured in (a) to a reference level (or reference levels),        thereby optimizing BNP-type peptide guided therapy.

The patient in accordance with the aforementioned method preferablydisplays a level (in particular a blood, serum or plasma level) of aBNP-type peptide which is below the reference level for said BNP-typepeptide being indicative of intensification of heart failure therapy.

The definitions given herein above apply mutatis mutandis to thefollowing embodiments of the present invention.

Methods for Predicting the Risk of Cardiac Decompensation,Hospitalization and/or Mortality

Further, the present invention is directed to a method, in particular anin vitro method, for predicting the risk of a patient who has heartfailure and who receives BNP-type peptide guided heart failure therapyto suffer from cardiac decompensation, hospitalization, and/or mortality(death) said method comprising the steps of

-   -   (a) measuring the level of at least one marker selected from the        group consisting of creatinine, urea, sodium, glucose, HbA1c        (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive        protein, in particular high sensitive CRP), Cystatin C, IL-6        (Interleukin 6), Prealbumin, sFlt-1 (soluble fms-like tyrosine        kinase-1), uric acid, GDF-15 (Growth Differentiation Factor 15),        Galectin-3 (Gal-3), Endostatin, Mimecan, IGFBP7 (Insulin Growth        Factor Binding Protein 7), sST2 (soluble ST2), and Osteopontin        in a sample from a patient who has heart failure and who        receives BNP-type peptide guided heart failure therapy, and    -   (b) comparing the level (or levels) of the marker (or markers)        measured in (a) to a reference level (or reference levels).

The method may further comprise the step of (c) predicting (or providinga prediction of) the risk of the patient to suffer from cardiacdecompensation, hospitalization, and/or mortality, in particular whereina level (levels) of the at least one marker above or below the referencelevel (levels) indicates that the patient has an increased risk tosuffer from cardiac decompensation, hospitalization, and/or mortality,and wherein a level (levels) of the at least one marker above or belowthe reference level (levels) indicates that the patient has a decreasedrisk to suffer from cardiac decompensation, hospitalization, and/ormortality.

In a preferred embodiment, the patient displays a level (in particular ablood, serum or plasma level) of a BNP-type peptide which is below thereference level for said BNP-type peptide being indicative ofintensification of heart failure therapy.

The following applies as diagnostic algorithm:

The following applies as diagnostic algorithm if the at least one markeris selected from the group consisting of creatinine, urea, glucose,HbA1c (glycated hemoglobin), CRP (C-reactive protein, in particular highsensitive CRP), Cystatin C, IL-6 (Interleukin 6), Prealbumin, sFlt-1(soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (GrowthDifferentiation Factor 15), Galectin-3 (Gal-3), Endostatin, Mimecan,sST2 (soluble ST2), and Osteopontin:

Preferably, a level (levels) of the at least one marker in the samplefrom the patient which is above the reference level (reference levels)for said marker (markers) indicates that the patient has an increasedrisk to suffer from cardiac decompensation, hospitalization, and/ormortality, and/or wherein a level (levels) of the at least one marker inthe sample from the patient which is below the reference level(reference levels) for said marker (markers) indicates that the patienthas a decreased risk to suffer from cardiac decompensation,hospitalization, and/or mortality.

The following applies as diagnostic algorithm if the at least one markeris selected from the group consisting of sodium, hemoglobin, hematocrit,and IGFBP-7:

Preferably, a level (levels) of the at least one marker in the samplefrom the patient which is below the reference level (reference levels)for said marker (markers) indicates that the patient has an increasedrisk to suffer from cardiac decompensation, hospitalization, and/ormortality, and/or wherein a level (levels) of the at least one marker inthe sample from the patient which is above the reference level(reference levels) for said marker (markers) indicates that the patienthas a decreased risk to suffer from cardiac decompensation,hospitalization, and/or mortality.

Preferred reference levels or ranges of reference levels are disclosedelsewhere herein (see Table A).

The phrase “providing a predication” as used herein refers to using theinformation or data generated relating to the level of the at least onebiomarker in a sample of the patient as referred to herein to predictthe risk of the patient to suffer from cardiac decompensation,hospitalization, and/or mortality. The information or data may be in anyform, written, oral or electronic. In some embodiments, using theinformation or data generated includes communicating, presenting,reporting, storing, sending, transferring, supplying, transmitting,dispensing, or combinations thereof. In some embodiments, communicating,presenting, reporting, storing, sending, transferring, supplying,transmitting, dispensing, or combinations thereof are performed by acomputing device, analyzer unit or combination thereof. In some furtherembodiments, communicating, presenting, reporting, storing, sending,transferring, supplying, transmitting, dispensing, or combinationsthereof are performed by a laboratory or medical professional. In someembodiments, the information or data includes a comparison of the levelof the at least one marker to a reference level. In some embodiments,the information or data includes an indication that the patient at riskor not at risk to suffer from cardiac decompensation, hospitalization,and/or mortality.

The present invention also relates to a method, in particular an invitro method for predicting the risk of a patient who has heart failureand who receives BNP-type peptide guided heart failure therapy to sufferfrom cardiac decompensation, hospitalization, and/or mortality (death),said method comprising the step of

-   -   (a) measuring the level of a BNP-type peptide in a in a sample        from a patient who has heart failure and who receives BNP-type        peptide guided heart failure therapy;    -   (b) measuring the level of at least one marker selected from the        group consisting of creatinine, urea, sodium, glucose, HbA1c        (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive        protein, in particular high sensitive CRP), Cystatin C, IL-6        (Interleukin 6), Prealbumin, sFlt-1 (soluble fms-like tyrosine        kinase-1), uric acid, GDF-15 (Growth Differentiation Factor 15),        Galectin-3 (Gal-3), Endostatin, Mimecan, IGFBP7 (Insulin Growth        Factor Binding Protein 7), sST2 (soluble ST2), and Osteopontin        in a sample from the patient,    -   (c) comparing the level of the BNP-type peptide measured in (a)        to a reference level (or reference levels), and    -   (d) comparing the level (or levels) of the at least one marker        measured in (b) to a reference level (or reference levels).

The method may further comprise the step of (f) predicting (or providinga prediction of) the risk of the patient to suffer from cardiacdecompensation, hospitalization, and/or mortality. The prediction ispreferably based on the results of the comparison steps.

The patient to be tested in accordance with the aforementioned methodmay display any level (in particular any blood, serum or plasma level)of a BNP-type peptide.

The following applies as diagnostic algorithm if the at least one markeras measured in step (b) is selected from the group consisting ofcreatinine, urea, glucose, HbA1c (glycated hemoglobin), CRP (C-reactiveprotein, in particular high sensitive CRP), Cystatin C, IL-6(Interleukin 6), Prealbumin, sFlt-1 (soluble fms-like tyrosinekinase-1), uric acid, GDF-15 (Growth Differentiation Factor 15),Galectin-3 (Gal-3), Endostatin, Mimecan, sST2 (soluble ST2), andOsteopontin:

-   -   (a) a level of the at least one marker in the sample from the        patient which is above the reference level for said marker, and        a level of BNP-type peptide which is above the reference level        for said BNP-type peptide is indicative for a patient who has an        increased risk to suffer from cardiac decompensation,        hospitalization, and/or mortality,    -   (b) a level of the at least one marker in the sample from the        patient which is above the reference level for said marker, and        a level of said BNP-type peptide which is below the reference        level for said BNP-type peptide is indicative for a patient who        has an increased risk to suffer from cardiac decompensation,        hospitalization, and/or mortality,    -   (c) a level of the at least one marker in the sample from the        patient which is below the reference level for said marker, and        a level of said BNP-type peptide which is above the reference        level for said BNP-type peptide is indicative for a patient who        has an increased risk to suffer from cardiac decompensation,        hospitalization, and/or mortality, and/or    -   (d) a level of the at least one marker in the sample from the        patient which is below the reference level for said marker, and        a level of said BNP-type peptide which is below the reference        level for said BNP-type peptide is indicative for a patient who        has a decreased risk to suffer from cardiac decompensation,        hospitalization, and/or mortality.

Alternatively or additionally, the following applies as diagnosticalgorithm if the at least one marker as measured in step (b) is selectedfrom the group consisting of sodium, hemoglobin, hematocrit, andIGFBP-7:

-   -   (a) a level of the at least one marker in the sample from the        patient which is below the reference level for said marker, and        a level of said BNP-type peptide which is above the reference        level for said BNP-type peptide is indicative for a patient who        has an increased risk to suffer from cardiac decompensation,        hospitalization, and/or mortality,    -   (b) a level of the at least one marker in the sample from the        patient which is below the reference level for said marker, and        a level of said BNP-type peptide which is below the reference        level for said BNP-type peptide is indicative for a patient who        has an increased risk to suffer from cardiac decompensation,        hospitalization, and/or mortality,    -   (c) a level of the at least one marker in the sample from the        patient which is above the reference level for said marker, and        a level of said BNP-type peptide which is above the reference        level for said BNP-type peptide is indicative for a patient who        has an increased risk to suffer from cardiac decompensation,        hospitalization, and/or mortality and/or    -   (d) a level of the at least one marker in the sample from the        patient which is above the reference level for said marker, and        a level of said BNP-type peptide which is below the reference        level for said BNP-type peptide is indicative for a patient who        has a decreased risk to suffer from cardiac decompensation,        hospitalization, and/or mortality.

Preferred reference levels or ranges of reference levels are disclosedelsewhere herein (see e.g. table A).

The term “cardiac decompensation” is well known in the art. Preferably,the term refers to a condition of chronic heart failure in which theheart is unable to ensure adequate cellular perfusion in all parts ofthe body without assistance. Accordingly, the compensatory mechanisms ofthe body are no longer sufficient to maintain pump function.

The term “mortality” as used herein relates to any kind of mortality, inparticular mortality which is caused by a cardiovascular complication.Preferably, said mortality is caused by heart failure.

The term “hospitalization” is well known in the art. As used herein,term relates to hospitalization which is caused by a cardiovascularcomplication. Preferably, said hospitalization is caused by heartfailure.

The term “predicting” used herein refers to assessing the probabilityaccording to which a patient as referred to herein will suffer fromcardiac decompensation, hospitalization, and/or mortality within adefined time window (predictive window) in the future. The predictivewindow is an interval in which the patient will develop cardiacdecompensation, will be hospitalized and/or will die according to thepredicted probability. The predictive window may be the entire remaininglifespan of the patient upon analysis by the method of the presentinvention. Preferably, however, the predictive window is an interval ofone, two, three, four, five, ten, fifteen or 20 years after the methodof the present invention has been carried out (more preferably andprecisely, after the sample to be analyzed by the method of the presentinvention has been obtained). Most preferably, said predictive window isan interval of four or five years. As will be understood by thoseskilled in the art, such an assessment is usually not intended to becorrect for 100% of the patients to be analyzed. The term, however,requires that the assessment will be valid for a statisticallysignificant portion of the patients to be analyzed. Whether a portion isstatistically significant can be determined without further ado by theperson skilled in the art using various well known statistic evaluationtools, e.g., determination of confidence intervals, p-valuedetermination, Student's t-test, Mann-Whitney test, etc. Details arefound in Dowdy and Wearden, Statistics for Research, John Wiley & Sons,New York 1983. Preferred confidence intervals are at least 90%, at least95%, at least 97%, at least 98% or at least 99%. The p-values are,preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, theprobability envisaged by the present invention allows that theprediction will be correct for at least 60%, at least 70%, at least 80%,or at least 90% of the patients of a given cohort.

The expression “predicting the risk of cardiac decompensation,hospitalization, or death” as used herein means that it the patient tobe analyzed by the method of the present invention is allocated eitherinto the group of patients of a population having an increased risk, orinto a group having a reduced risk. An increased risk as referred to inaccordance with the present invention, preferably, means that the riskof developing cardiac decompensation, of hospitalization, or ofmortality within a predetermined predictive window is increasedsignificantly (i.e. increased significantly) for a patient with respectto the average risk for a such an event in a population of patientshaving heart failure and receiving BNP-type peptide guided therapy. Areduced risk as referred to in accordance with the present invention,preferably, means that the risk of developing cardiac decompensation, ofhospitalization, or of mortality within a predetermined predictivewindow is reduced significantly for a patient with respect to theaverage risk for such an event in a population of said patients.Particularly, a significant increase or reduction of a risk is anincrease or reduction or a risk of a size which is considered to besignificant for prognosis, particularly said increase or reduction isconsidered statistically significant. The terms “significant” and“statistically significant” are known by the person skilled in the art.Thus, whether an increase or reduction of a risk is significant orstatistically significant can be determined without further ado by theperson skilled in the art using various well known statistic evaluationtools.

Preferably, for a predictive window of tree years, an increased risk iswithin the range of 3.0% and 19.0%, more preferably within the range of12.0% to 17.0%, most preferably, within the range of 8.0% to 16.0%. Anincreased, and, thus increased risk as used herein, preferably, relatesto a risk of more than 3.0%, preferably, more than 12.0%, morepreferably, more than 17%, even more preferably, more than 20%,preferably, with respect to a predictive window of three years. Areduced risk as used herein, preferably, relates to a risk of less than8.0%, preferably, less than 6%, even more preferably, less than 4%, andis, most preferably within the range of 3.0% and 8.0%, preferably withrespect to a predictive window of three years.

The present invention also relates to the use of at least one markerselected from the group consisting of creatinine, urea, sodium, glucose,HbA1c (glycated hemoglobin), CRP (C-reactive protein, in particular highsensitive CRP), Cystatin C, IL-6 (Interleukin 6), Prealbumin, sFlt-1(soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (GrowthDifferentiation Factor 15), Galectin-3 (Gal-3), Endostatin, Mimecan,sST2 (soluble ST2), and Osteopontin, and/or the use of at least onedetection agent for said at least one marker (i.e. for creatinine, urea,sodium, glucose, HbA1c (glycated hemoglobin), CRP (C-reactive protein,in particular high sensitive CRP), Cystatin C, IL-6 (Interleukin 6),Prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid,GDF-15 (Growth Differentiation Factor 15), Galectin-3 (Gal-3),Endostatin, Mimecan, sST2 (soluble ST2), or Osteopontin) in a sample ofa patient having heart failure and receiving BNP-type peptide guidedheart failure therapy for identifying a patient being eligible tointensification of heart failure therapy, for predicting the risk of thepatient of suffering from cardiac decompensation, of hospitalization,and or of mortality, or for optimizing BNP-type peptide guided heartfailure therapy.

The present invention also relates to the i) use of the QRS duration,optionally in combination with a BNP-type peptide, and/or the use of adevice for determining the QRS duration such as an ECG device (i.e. adevice which can generate an electrocardiogram), optionally incombination with at least one detection agent for a BNP-type peptide,for identifying a patient being eligible to intensification of heartfailure therapy, for predicting the risk of the patient of sufferingfrom cardiac decompensation, of hospitalization, and or of mortality, orfor optimizing BNP-type peptide guided heart failure therapy. Asoutlined elsewhere herein, the patient shall receive BNP-type peptideguided heart failure therapy.

The present invention also relates to the use of a BNP-type peptide incombination with at least one marker selected from the group consistingof creatinine, urea, sodium, glucose, HbA1c (glycated hemoglobin), CRP(C-reactive protein, in particular high sensitive CRP), Cystatin C, IL-6(Interleukin 6), Prealbumin, sFlt-1 (soluble fms-like tyrosinekinase-1), uric acid, GDF-15 (Growth Differentiation Factor 15),Galectin-3 (Gal-3), Endostatin, Mimecan, sST2 (soluble ST2), andOsteopontin, and/or the use of an detection agent which specificallybinds to a BNP-type peptide in combination of at least one detectionagent for a marker selected from the group of marker selected from thegroup consisting of creatinine, urea, sodium, glucose, HbA1c (glycatedhemoglobin), CRP (C-reactive protein, in particular high sensitive CRP),Cystatin C, IL-6 (Interleukin 6), Prealbumin, sFlt-1 (soluble fms-liketyrosine kinase-1), uric acid, GDF-15 (Growth Differentiation Factor15), Galectin-3 (Gal-3), Endostatin, Mimecan, sST2 (soluble ST2), andOsteopontin, in a sample of a patient having heart failure and receivingBNP-type peptide guided heart failure therapy for identifying a patientbeing eligible to intensification of heart failure therapy, or forpredicting the risk of the patient of suffering from cardiacdecompensation, of hospitalization, or of mortality.

The present invention further relates to the use of at least one markerselected from the group consisting of creatinine, urea, sodium, glucose,HbA1c (glycated hemoglobin), hemoglobin, hematocrit, IGFBP7, CRP(C-reactive protein, in particular high sensitive CRP), Cystatin C, IL-6(Interleukin 6), Prealbumin, sFlt-1 (soluble fms-like tyrosinekinase-1), uric acid, GDF-15 (Growth Differentiation Factor 15),Galectin-3 (Gal-3), Endostatin, Mimecan, sST2 (soluble ST2), andOsteopontin, and/or the use of at least one detection agent for said atleast one marker (i.e. for creatinine, urea, sodium, glucose, HbA1c(glycated hemoglobin), hemoglobin, hematocrit, IGFBP7, CRP (C-reactiveprotein, in particular high sensitive CRP), Cystatin C, IL-6(Interleukin 6), Prealbumin, sFlt-1 (soluble fms-like tyrosinekinase-1), uric acid, GDF-15 (Growth Differentiation Factor 15),Galectin-3 (Gal-3), Endostatin, Mimecan, sST2 (soluble ST2), orOsteopontin) for the manufacture of a diagnostic composition foridentifying a patient being eligible to intensification of heart failuretherapy, or for predicting the risk of the patient of suffering fromcardiac decompensation, of hospitalization, or of mortality (inparticular, in a sample of a patient having heart failure and receivingBNP-type peptide guided heart failure therapy).

The present invention also relates to the use of a BNP-type peptide incombination with at least one marker selected from the group consistingof creatinine, urea, sodium, glucose, HbA1c (glycated hemoglobin),hemoglobin, hematocrit, IGFBP7, CRP (C-reactive protein, in particularhigh sensitive CRP), Cystatin C, IL-6 (Interleukin 6), Prealbumin,sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (GrowthDifferentiation Factor 15), Galectin-3 (Gal-3), Endostatin, Mimecan,sST2 (soluble ST2), and Osteopontin, and/or the use of an detectionagent which specifically binds to a BNP-type peptide in combination withat least one detection agent for a marker selected from the group ofmarker selected from the group consisting of creatinine, urea, sodium,glucose, HbA1c (glycated hemoglobin), hemoglobin, hematocrit, IGFBP7,CRP (C-reactive protein, in particular high sensitive CRP), Cystatin C,IL-6 (Interleukin 6), Prealbumin, sFlt-1 (soluble fms-like tyrosinekinase-1), uric acid, GDF-15 (Growth Differentiation Factor 15),Galectin-3 (Gal-3), Endostatin, Mimecan, sST2 (soluble ST2), andOsteopontin, for the manufacture of a diagnostic composition foridentifying a patient being eligible to intensification of heart failuretherapy, or for predicting the risk of the patient of suffering fromcardiac decompensation, of hospitalization, or of mortality (inparticular, in a sample of a patient having heart failure and receivingBNP-type peptide guided heart failure therapy).

If the marker is a polypeptide or peptide, in particular if the markeris HbA1c (glycated hemoglobin), CRP (C-reactive protein, in particularhigh sensitive CRP), Cystatin C, IL-6 (Interleukin 6), Prealbumin,sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (GrowthDifferentiation Factor 15), Galectin-3 (Gal-3), Endostatin, Mimecan,sST2 (soluble ST2), and Osteopontin, the detection agent preferablyspecifically binds to said marker. In this case, the detection agent ispreferably a monoclonal or polyclonal antibody (for a definition of theterm “antibody” see elsewhere herein). For the remaining markers, thedetection agent may be an agent that forms an complex with the markerthereby allowing the measurement of the level of the marker, or enzymethat allows for the conversion of the marker as described elsewhereherein.

If the marker is creatinine, the detection agent may be picric acidwhich forms a complex with creatinine.

If the marker is uric acid, the detection agent may be uricase orperoxidase.

If the marker is urea, the detection agent may be urease.

If the marker is glucose, the detection agent may be a hexokinase.

According, the present invention also preferably relates to a system foridentifying a patient who is eligible to an intensification of heartfailure therapy, comprising

-   -   a) an analyzer unit configured to contact, in vitro, a portion        of a sample from the patient with a detection agent (or agents        if the level of at least one marker is measured) for measuring        the level of at least one marker selected from the group        consisting of creatinine, urea, sodium, glucose, HbA1c (glycated        hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, in        particular high sensitive CRP), Cystatin C, IL-6 (Interleukin        6), Prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1),        uric acid, GDF-15 (Growth Differentiation Factor 15), Galectin-3        (Gal-3), Endostatin, Mimecan, IGFBP7 (Insulin Growth Factor        Binding Protein 7), sST2 (soluble ST2), and Osteopontin,    -   b) an analyzer unit configured to detect a signal from the        portion of the sample from the patient contacted with the agent        (or agents),    -   c) a computing device having a processor and in operable        communication with said analysis units, and    -   d) a non-transient machine readable media including a plurality        of instruction executable by a the processor, the instructions,        when executed calculate a level of the at least one marker, and        compare the level of the at least one marker with a reference        level (or reference levels if the level of more than one marker        is measured), thereby identifying a patient who is eligible to        an intensification of heart failure therapy.

As set forth above, the patient shall have heart failure and shallreceive BNP-type peptide guided heart failure therapy.

Furthermore, a device adapted for carrying out the method of the presentinvention is provided, said device comprising

-   -   a) an analyzer unit comprising a detection agent (or agents) for        measuring the level of at least one marker selected from the        group consisting of creatinine, urea, sodium, glucose, HbA1c        (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive        protein, in particular high sensitive CRP), Cystatin C, IL-6        (Interleukin 6), Prealbumin, sFlt-1 (soluble fms-like tyrosine        kinase-1), uric acid, GDF-15 (Growth Differentiation Factor 15),        Galectin-3 (Gal-3), Endostatin, Mimecan, IGFBP7 (Insulin Growth        Factor Binding Protein 7), sST2 (soluble ST2), and Osteopontin        in a sample of a patient who has heart failure and who receives        BNP-type peptide guided heart failure therapy, and    -   b) an analyzer unit for comparing the measured level(s) with        reference level(s), whereby a patient is identified who is        eligible to an intensification of heart failure therapy        antagonist, a diuretic, and an inhibitor of the        renin-angiotensin system, said unit comprising a database with a        reference level (or levels) and a computer-implemented algorithm        carrying out the comparison.

Preferred reference levels and diagnostic algorithms are disclosedelsewhere herein.

A preferred embodiment of the instant disclosure includes a system foridentifying a subject being eligible to the administration of at leastone medicament selected from the group consisting of a beta blocker, analdosterone antagonist, a diuretic, and an inhibitor of therenin-angiotensin system. Examples of systems include clinical chemistryanalyzers, coagulation chemistry analyzers, immunochemistry analyzers,urine analyzers, nucleic acid analyzers, used to detect the result ofchemical or biological reactions or to monitor the progress of chemicalor biological reactions. More specifically, exemplary systems of theinstant disclosure may include Roche Elecsys™ Systems and Cobas® eImmunoassay Analyzers, Abbott Architect™ and Axsym™ Analyzers, SiemensCentaur™ and Immulite™ Analyzers, and Beckman Coulter UniCel™ and Acess™Analyzers, or the like.

Embodiments of the system may include one or more analyzer unitsutilized for practicing the subject disclosure. The analyzer units ofthe system disclosed herein are in operable communication with thecomputing device disclosed herein through any of a wired connection,Bluetooth, LANS, or wireless signal, as are known. Additionally,according to the instant disclosure, an analyzer unit may comprise astand-alone apparatus, or module within a larger instrument, whichperforms one or both of the detection, e.g. qualitative and/orquantitative evaluation of samples for diagnostic purpose. For example,an analyzer unit may perform or assist with the pipetting, dosing,mixing of samples and/or reagents. An analyzer unit may comprise areagent holding unit for holding reagents to perform the assays.Reagents may be arranged for example in the form of containers orcassettes containing individual reagents or group of reagents, placed inappropriate receptacles or positions within a storage compartment orconveyor. Detection reagents may also be in immobilized form on a solidsupport which are contacted with the sample. Further, an analyzer unitmay include a process and/or detection component which is optimizablefor specific analysis.

According to some embodiments, an analyzer unit may be configured foroptical detection of an analyte, for example a marker, with a sample. Anexemplary analyzer unit configured for optical detection comprises adevice configured for converting electro-magnetic energy into anelectrical signal, which includes both single and multi-element or arrayoptical detectors. According to the present disclosure, an opticaldetector is capable of monitoring an optical electro-magnetic signal andproviding an electrical outlet signal or response signal relative to abaseline signal indicative of the presence and/or concentration of ananalyte in a sample being located in an optical path. Such devices mayalso include, for example, photodiodes, including avalanche photodiodes,phototransistors, photoconductive detectors, linear sensor arrays, CCDdetectors, CMOS detectors, including CMOS array detectors,photomultipliers, and photomultiplier arrays. According to certainembodiments, an optical detector, such as a photodiode orphotomultiplier, may contain additional signal conditioning orprocessing electronics. For example, an optical detector may include atleast one pre-amplifier, electronic filter, or integrated circuit.Suitable pre-preamplifiers include, for example, integrating,transimpedance, and current gain (current mirror) pre-amplifiers.

Additionally, one or more analyzer unit according to the instantdisclosure may comprise a light source for emitting light. For example,a light source of an analyzer unit may consist of at least one lightemitting element (such as a light emitting diode, an electric poweredradiation source such as an incandescent lamp, an electroluminescentlamp, a gas discharge lamp, a high-intensity discharge lamp, a laser)for measuring analyte concentrations with a sample being tested or forenabling an energy transfer (for example, through florescent resonanceenergy transfer or catalyzing an enzyme).

Further, an analyzer unit of the system may include one or moreincubation units (for example, for maintaining a sample or a reagent ata specified temperature or temperature range). In some embodiments, ananalyzer unit may include a thermocycler, include a real-timethermocycler, for subjecting a sample to repeated temperature cycles andmonitoring a change in the level of an amplification product with thesample.

Additionally, an analyzer unit of the system disclosed herein maycomprise, or be operationally connected to, a reaction vessel or cuvettefeeding unit. Exemplary feeding units include liquid processing units,such as a pipetting unit, to deliver samples and/or reagents to thereaction vessels. The pipetting unit may comprise a reusable washableneedle, e.g. a steel needle, or disposable pipette tips. The analyzerunit may further comprise one or more mixing units, for example a shakerto shake a cuvette comprising a liquid, or a mixing paddle to mixliquids in a cuvette, or reagent container.

It follows from the above that according to some embodiments of theinstant disclosure, portions of some steps of methods disclosed anddescribed herein may be performed by a computing device. A computingdevice may be a general purpose computer or a portable computing device,for example. It should also be understood that multiple computingdevices may be used together, such as over a network or other methods oftransferring data, for performing one or more steps of the methodsdisclosed herein. Exemplary computing devices include desktop computers,laptop computers, personal data assistants (“PDA”), such as BLACKBERRYbrand devices, cellular devices, tablet computers, servers, and thelike. In general, a computing device comprises a processor capable ofexecuting a plurality of instructions (such as a program of software).

A computing device has access to a memory. A memory is a computerreadable medium and may comprise a single storage device or multiplestorage devices, located either locally with the computing device oraccessible to the computing device across a network, for example.Computer-readable media may be any available media that can be accessedby the computing device and includes both volatile and non-volatilemedia. Further, computer readable-media may be one or both of removableand non-removable media. By way of example, and not limitation,computer-readable media may comprise computer storage media. Exemplarycomputer storage media includes, but is not limited to, RAM, ROM,EEPROM, flash memory or any other memory technology, CD-ROM, DigitalVersatile Disk (DVD) or other optical disk storage, magnetic cassettes,magnetic tape, magnetic disk storage or other magnetic storage devices,or any other medium which can be used for storing a plurality ofinstructions capable of being accessed by the computing device andexecuted by the processor of the computing device.

According to embodiments of the instant disclosure, software may includeinstructions which, when executed by a processor of the computingdevice, may perform one or more steps of the methods disclosed herein.Some of the instructions may be adapted to produce signals that controloperation of other machines and thus may operate through those controlsignals to transform materials far removed from the computer itself.These descriptions and representations are the means used by thoseskilled in the art of data processing, for example, to most effectivelyconvey the substance of their work to others skilled in the art.

The plurality of instructions may also comprise an algorithm which isgenerally conceived to be a self-consistent sequence of steps leading toa desired result. These steps are those requiring physical manipulationsof physical quantities. Usually, though not necessarily, thesequantities take the form of electrical or magnetic pulses or signalscapable of being stored, transferred, transformed, combined, compared,and otherwise manipulated. It proves convenient at times, principallyfor reasons of common usage, to refer to these signals as values,characters, display data, numbers, or the like as a reference to thephysical items or manifestations in which such signals are embodied orexpressed. It should be borne in mind, however, that all of these andsimilar terms are to be associated with the appropriate physicalquantities and are merely used here as convenient labels applied tothese quantities. According to some embodiments of the instantdisclosure, an algorithm for carrying out a comparison between adetermined level of one or more markers disclosed herein, and a suitablereference, is embodied and performed by executing the instructions. Theresults may be given as output of parametric diagnostic raw data or asabsolute or relative levels. According to various embodiments of thesystem disclosed herein, a “diagnosis” may be provided by the computingdevice of a system disclosed herein based on said comparison of thecalculated “level” to a reference or a threshold. For example, acomputing device of a system may provide an indicator, in the form of aword, symbol, or numerical value which is indicative of a particulardiagnosis.

The computing device may also have access to an output device. Exemplaryoutput devices include fax machines, displays, printers, and files, forexample. According to some embodiments of the present disclosure, acomputing device may perform one or more steps of a method disclosedherein, and thereafter provide an output, via an output device, relatingto a result, indication, ratio or other factor of the method.

Finally, the invention pertains to a kit adapted for carrying out amethod of the present invention comprising at least a detection agent(or agents if the level of at least one marker is measured) formeasuring the level of at least one marker selected from the groupconsisting of creatinine, urea, sodium, glucose, HbA1c (glycatedhemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, inparticular high sensitive CRP), Cystatin C, IL-6 (Interleukin 6),Prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid,GDF-15 (Growth Differentiation Factor 15), Galectin-3 (Gal-3),Endostatin, Mimecan, IGFBP7 (Insulin Growth Factor Binding Protein 7),sST2 (soluble ST2), and Osteopontin, reference standards as well asinstructions for carrying out the said method.

The term “kit” as used herein refers to a collection of theaforementioned components, preferably, provided in separately or withina single container. The container also comprises instructions forcarrying out the method of the present invention. These instructions maybe in the form of a manual or may be provided by a computer program codewhich is capable of carrying out the comparisons referred to in themethods of the present invention and to establish a diagnosisaccordingly when implemented on a computer or a data processing device.The computer program code may be provided on a data storage medium ordevice such as a optical storage medium (e.g., a Compact Disc) ordirectly on a computer or data processing device. Further, the kit shallcomprise at least one standard for a reference as defined herein above,i.e. a solution with a pre-defined level for the biomarker as referredto herein representing a reference level.

In some embodiments, a kit disclosed herein includes at least onecomponent or a packaged combination of components for practicing adisclosed method. By “packaged combination” it is meant that the kitsprovide a single package that contains a combination of one or morecomponents, such as probes (for example, an antibody), controls,buffers, reagents (for example, conjugate and/or substrate)instructions, and the like, as disclosed herein. A kit containing asingle container is also included within the definition of “packagedcombination.” In some embodiments, the kits include at least one probe,for example an antibody (having specific affinity for an epitope of abiomarker as disclosed herein. For example, the kits may include anantibody that is labelled with a fluorophore or an antibody that is amember of a fusion protein. In the kit, the probe may be immobilized,and may be immobilized in a specific conformation. For example, animmobilized probe may be provided in a kit to specifically bind targetprotein, to detect target protein in a sample, and/or to remove targetprotein from a sample.

According to some embodiments, kits include at least one probe, whichmay be immobilized, in at least one container. Kits may also includemultiple probes, optionally immobilized, in one or more containers. Forexample, the multiple probes may be present in a single container or inseparate containers, for example, wherein each container contains asingle probe.

In some embodiments, a kit may include one or more non-immobilized probeand one or more solid support that does or does not include animmobilized probe. Some such embodiments may comprise some or all of thereagents and supplies needed for immobilizing one or more probes to thesolid support, or some or all of the reagents and supplies needed forbinding of immobilized probes to specific proteins within a sample.

In certain embodiments, a single probe (including multiple copies of thesame probe) may be immobilized on a single solid support and provided ina single container. In other embodiments, two or more probes, eachspecific for a different target protein or a different form of a singletarget protein (such as a specific epitope), a provided in a singlecontainer. In some such embodiments, an immobilized probe may beprovided in multiple different containers (e.g., in single-use form), ormultiple immobilized probes may be provided in multiple differentcontainers. In further embodiments, the probes may be immobilized onmultiple different type of solid supports. Any combination ofimmobilized probe(s) and container(s) is contemplated for the kitsdisclosed herein, and any combination thereof may be selected to achievea suitable kit for a desired use.

A container of the kits may be any container that is suitable forpackaging and/or containing one or more components disclosed herein,including for example probes (for example, an antibody), controls,buffers, and reagents (for example, conjugate and/or substrate).Suitable materials include, but are not limited to, glass, plastic,cardboard or other paper product, wood, metal, and any alloy thereof. Insome embodiments, the container may completely encase an immobilizedprobe(s) or may simply cover the probe to minimize contamination bydust, oils, etc., and expose to light. In some further embodiments, hekits may comprise a single container or multiple containers, and wheremultiple containers are present, each container may be the same as allother containers, different than others, or different than some but notall other containers.

PREFERRED EMBODIMENTS OF THE PRESENT INVENTION

In the following, preferred embodiments of the present invention aredisclosed. The definitions and explanations given herein above and inthe claims apply mutatis mutandis.

-   1. A method for identifying a patient who is eligible to an    intensification of heart failure therapy, said method comprising the    steps of    -   (a) measuring the level of at least one marker selected from the        group consisting of creatinine, urea, sodium, glucose, HbA1c        (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive        protein, in particular high sensitive CRP), Cystatin C, IL-6        (Interleukin 6), Prealbumin, sFlt-1 (soluble fms-like tyrosine        kinase-1), uric acid, GDF-15 (Growth Differentiation Factor 15),        Galectin-3 (Gal-3), Endostatin, Mimecan, IGFBP7 (Insulin Growth        Factor Binding Protein 7), sST2 (soluble ST2), and Osteopontin        in a sample from a patient who has heart failure and who        receives BNP-type peptide guided heart failure therapy, and    -   (b) comparing the level (or levels) of the marker (or markers)        measured in (a) to a reference level (or reference levels).-   2. The method according to embodiment 1, further comprising step (c)    of identifying a patient who is eligible to an intensification of    heart failure therapy, or not.-   3. The method of embodiment 1 or 2, wherein the patient displays a    level of a BNP-type peptide which is below the reference level for    said BNP-type peptide being indicative of intensification of heart    failure therapy.-   4. The method according to any one of embodiments 1 to 4, wherein    -   i) the at least one marker is selected from the group consisting        of creatinine, urea, glucose, HbA1c (glycated hemoglobin), CRP        (C-reactive protein, in particular high sensitive CRP), Cystatin        C, IL-6 (Interleukin 6), Prealbumin, sFlt-1 (soluble fms-like        tyrosine kinase-1), uric acid, GDF-15 (Growth Differentiation        Factor 15), Galectin-3 (Gal-3), Endostatin, Mimecan, sST2        (soluble ST2), and Osteopontin, and wherein a level (levels) of        the at least one marker in the sample from the patient which is        above the reference level (reference levels) for said marker        (markers) indicates that the patient is eligible to        intensification of heart failure therapy, and/or wherein a level        (levels) of the at least one marker in the sample from the        patient which is below the reference level (reference levels)        for said marker (markers) indicates that the patient is not        eligible to intensification of heart failure therapy, and/or    -   ii) the at least one marker is selected from the group        consisting of sodium, hemoglobin, hematocrit, and IGFBP-7, and        wherein a level (levels) of the at least one marker in the        sample from the patient which is below the reference level        (reference levels) for said marker (markers) indicates that the        patient is eligible to intensification of heart failure therapy,        and/or wherein a level (levels) of the at least one marker in        the sample from the patient which is above the reference level        (reference levels) for said marker (markers) indicates that the        patient is not eligible to intensification of heart failure        therapy.-   5. A method for identifying a patient who is eligible to an    intensification of heart failure therapy, said method comprising the    step of    -   (a) measuring the level of a BNP-type peptide in a sample from a        patient who has heart failure and who receives BNP-type peptide        guided heart failure therapy;

(b) measuring the level of at least one marker selected from the groupconsisting of creatinine, urea, sodium, glucose, HbA1c (glycatedhemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, inparticular high sensitive CRP), Cystatin C, IL-6 (Interleukin 6),Prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid,GDF-15 (Growth Differentiation Factor 15), Galectin-3 (Gal-3),Endostatin, Mimecan, IGFBP7 (Insulin Growth Factor Binding Protein 7),sST2 (soluble ST2), and Osteopontin in a sample from the patient,

-   -   (c) comparing the level of the BNP-type peptide measured in (a)        to a reference level (or reference levels), and    -   (d) comparing the level (or levels) of the at least one marker        measured in (b) to a reference level (or reference levels).

-   6. The method according to embodiment 5, wherein    -   i) the at least one marker is selected from the group consisting        of creatinine, urea, glucose, HbA1c (glycated hemoglobin), CRP        (C-reactive protein, in particular high sensitive CRP), Cystatin        C, IL-6 (Interleukin 6), Prealbumin, sFlt-1 (soluble fms-like        tyrosine kinase-1), uric acid, GDF-15 (Growth Differentiation        Factor 15), Galectin-3 (Gal-3), Endostatin, Mimecan, sST2        (soluble ST2), and Osteopontin, and wherein        -   (a) a level of the at least one marker in the sample from            the patient which is above the reference level for said            marker, and a level of BNP-type peptide which is above the            reference level for said BNP-type peptide is indicative for            a patient who is eligible to intensification of heart            failure therapy,        -   (b) a level of the at least one marker in the sample from            the patient which is above the reference level for said            marker, and a level of said BNP-type peptide which is below            the reference level for said BNP-type peptide is indicative            for a patient who is eligible to intensification of heart            failure therapy,        -   (c) a level of the at least one marker in the sample from            the patient which is below the reference level for said            marker, and a level of said BNP-type peptide which is above            the reference level for said BNP-type peptide is indicative            for a patient who is eligible to intensification of heart            failure therapy, and/or        -   (d) a level of the at least one marker in the sample from            the patient which is below the reference level for said            marker, and a level of said BNP-type peptide which is below            the reference level for said BNP-type peptide is indicative            for a patient who is not eligible to intensification of            heart failure therapy, and/or    -   ii) the at least one marker is selected from the group        consisting of sodium, hemoglobin, hematocrit, and IGFBP-7,        -   (a) a level of the at least one marker in the sample from            the patient which is below the reference level for said            marker, and a level of said BNP-type peptide which is above            the reference level for said BNP-type peptide is indicative            for a patient who is eligible to intensification of heart            failure therapy,        -   (b) a level of the at least one marker in the sample from            the patient which is below the reference level for said            marker, and a level of said BNP-type peptide which is below            the reference level for said BNP-type peptide is indicative            for a patient who is eligible to intensification of heart            failure therapy,        -   (c) a level of the at least one marker in the sample from            the patient which is above the reference level for said            marker, and a level of said BNP-type peptide which is above            the reference level for said BNP-type peptide is indicative            for a patient who is eligible to intensification of heart            failure therapy, and/or        -   (d) a level of the at least one marker in the sample from            the patient which is above the reference level for said            marker, and a level of said BNP-type peptide which is below            the reference level for said BNP-type peptide is indicative            for a patient who is not eligible to intensification of            heart failure therapy.

-   7. A method for optimizing BNP-type peptide guided heart failure    therapy, said method comprising the steps of    -   (a) measuring the level of at least one marker selected from the        group consisting of creatinine, urea, sodium, glucose, HbA1c        (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive        protein, in particular high sensitive CRP), Cystatin C, IL-6        (Interleukin 6), Prealbumin, sFlt-1 (soluble fms-like tyrosine        kinase-1), uric acid, GDF-15 (Growth Differentiation Factor 15),        Galectin-3 (Gal-3), Endostatin, Mimecan, IGFBP7 (Insulin Growth        Factor Binding Protein 7), sST2 (soluble ST2), and Osteopontin        in a sample from a patient who has heart failure and who        receives BNP-type peptide guided therapy, and    -   (b) comparing the level (or levels) of the marker (or markers)        measured in (a) to a reference level (or reference levels),        thereby optimizing BNP-type peptide guided therapy.

-   8. The method according to any one of embodiments 1 to 7, wherein    the patient is human.

-   9. The method according to any one of embodiments to 1 to 8, wherein    the patient has heart failure classified as stage B or C according    to the ACC/AHA classification, and/or wherein the patient has heart    failure according to class II or III of the NYHA classification.

-   10. The method according to any of embodiments 1 to 9, wherein the    sample is a blood, serum or plasma sample.

-   11. The method according to any one of embodiments 1 to 10, wherein    the heart failure therapy is medicinal heart failure therapy, in    particular wherein the heart failure therapy comprises    administration of at least one medicament selected from the group    consisting of diuretics, angiotensin converting enzyme inhibitors,    angiotensin II receptor blockers, beta blockers and aldosterone    antagonists.

-   12. The method of embodiment 11, wherein the heart failure therapy    comprises combined administration of a beta blocker and an ACE    inhibitor.

-   13. The method according to any one of embodiments 1 to 12, wherein    the intensification of heart failure therapy comprises increasing    the dosage of previously administered medicaments, the    administration of a further medicament (or medicaments), in    particular the administration of a further medicament (or    medicaments) having a different mode of action that the previously    administered medicaments, device therapy, life style changes, and    combinations thereof.

-   14. Use of i) at least one marker selected from the group consisting    of creatinine, urea, sodium, glucose, HbA1c (glycated hemoglobin),    hemoglobin, hematocrit, IGFBP7, CRP (C-reactive protein, in    particular high sensitive CRP), Cystatin C, IL-6 (Interleukin 6),    Prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid,    GDF-15 (Growth Differentiation Factor 15), Galectin-3 (Gal-3),    Endostatin, Mimecan, sST2 (soluble ST2), and Osteopontin, and/or use    of ii) at least one detection agent for said at least one marker in    a sample of a patient having heart failure and receiving BNP-type    peptide guided heart failure therapy for identifying a patient being    eligible to intensification of heart failure therapy, for predicting    the risk of the patient of suffering from cardiac decompensation, of    hospitalization, and/or of mortality, or for optimizing BNP-type    peptide guided heart failure therapy.

-   15. Use of a BNP-type peptide in combination with at least one    marker selected from the group consisting of creatinine, urea,    sodium, glucose, HbA1c (glycated hemoglobin), hemoglobin,    hematocrit, IGFBP7, CRP (C-reactive protein, in particular high    sensitive CRP), Cystatin C, IL-6 (Interleukin 6), Prealbumin, sFlt-1    (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (Growth    Differentiation Factor 15), Galectin-3 (Gal-3), Endostatin, Mimecan,    sST2 (soluble ST2), and Osteopontin, and/or the use of an detection    agent which specifically binds to a BNP-type peptide in combination    with at least one detection agent for a marker selected from the    group of marker selected from the group consisting of creatinine,    urea, sodium, glucose, HbA1c (glycated hemoglobin), hemoglobin,    hematocrit, IGFBP7, CRP (C-reactive protein, in particular high    sensitive CRP), Cystatin C, IL-6 (Interleukin 6), Prealbumin, sFlt-1    (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (Growth    Differentiation Factor 15), Galectin-3 (Gal-3), Endostatin, Mimecan,    sST2 (soluble ST2), and Osteopontin, in a sample of a patient having    heart failure and receiving BNP-type peptide guided heart failure    therapy for identifying a patient being eligible to intensification    of heart failure therapy, or for predicting the risk of the patient    of suffering from cardiac decompensation, of hospitalization, or of    death.

All references referred to above are herewith incorporated by referencewith respect to their entire disclosure content as well as theirspecific disclosure content explicitly referred to in the abovedescription.

EXAMPLES

The invention will now be illustrated by the following Examples whichare not intended to restrict or limit the scope of this invention.

Example 1 Patients

499 patients suffering from HF (NYHA class II-IV systolic HF (LVEF ≦45%)were guided according to NT-proBNP target or usual care (Pfisterer M. etal. JAMA. 2009; 301:383-92). Overall, patients with NT-proBNP levels<1000 pg/ml, a previously identified cut-off value for good outcome, hadsignificantly better outcome than those with NT-proBNP levels that couldnot be reduced to these levels. However, some of the patients with lowNT-proBNP levels <1000 pg/mL remained at risk. Additionally, somepatients with NT-proBNP levels slightly above 1000 pg/mL were atunexpectedly high risk. This risk could be identified with good accuracyby additional measurement of marker and clinical parameter levels, forexample after 6 months of therapy. Moreover, these additional markersand parameters also provided additional important information forpotential therapy guidance in the group with higher risk (i.e. NT-proBNPlevels after 6 months >1000 pg/ml).

BNP and/or NT-proBNP levels together with one or several markers and/orclinical parameters are measured at regular intervals every few weeks upto every six months. If intensification of medical therapy is eitherclinically necessary and/or indicated by the BNP/NT-proBNP and/or one ofthese additional markers and/or parameters, these subjects makefollow-up visits in the clinic every few weeks until either anoptimal/maximal medical therapy is achieved, the BNP/NT-proBNP targetgoal of ≦100-200 pg/mL and ≦1,000 pg/mL, respectively, is reached, andthe following target goals or the subject requires hospitalization.

Table 1 shows cutoffs for additional markers and clinical parameters(based on ROC-optimized cutoffs and inflection points of risk deciles;both methods render similar cutoffs for identification of residual risk;when both are available the lower cutoff should be applied):

TABLE 1 Parameter Marker Units ROC optimized cutoff Risk decile cutoffCreatinine mg/dL 1.5 Not applicable (n.a) BUN (urea) mmol/L 12.1 11.1Glucose mmol/L n.a. 11.6 HbA1c % 0.06 n.a. hsCRP mg/mL 10.4 12.7Cystatin C mg/L 1.9 n.a. IL-6 pg/mL 9.3 9.9 Prealbumin g/L 0.16 n.a.sFLt-1 pg/mL 87.3 94.3 Uric Acid mg/dL 9.1 9.4 GFD-15 pg/mL 4270 3210sST2 ng/mL 41.5 45.0 Galectin-3 ng/mL 29.0 25.1 Endostatin ng/mL 243 257Mimecan ng/mL 45.3 46.7 IGFBP-7 ng/mL 75.3 71.4 Osteopontin ng/mL 113.5114.5 Hemoglobin g/dL 8.04 n.a. Hematocrit % 0.40 n.a. QRS duration ms159 160 Sodium mmol/L 140.5 141

Table 2 showing Wald scores, p-values and Hazard Ratios (HR, with 95%confidence intervals) of biomarkers and clinical parameters in patientsguided according to NT-proBNP. Wald scores and Hazard Ratio indicateremaining risk of decompensation, hospitalization, or death in patientsguided with BNP-type peptides.

variable Wald P-value HR 95% CI Hb 2.0 .16 .82  .62-1.08 creatinine 19.9<.001 3.76 2.10-6.73 BUN 24.1 <.001 1.10 1.06-1.14 Hct 2.0 .16 .01 .00-5.80 Glucose 3.5 .06 1.11 1.00-1.23 HbA1c 3.3 .07 n/a n/a hsCRP log4.3 .04 1.68 1.03-2.75 CysC 17.8 <.001 2.71 1.71-4.31 IL6 log 7.7 .0062.64 1.33-5.24 PREA BL 5.5 .02 .001 .00-.32 sFIT log 3.0 .08 3.20 .86-11.84 hsTnT log 15.0 <.001 3.81 1.94-7.48 uric acid 5.0 .03 1.191.02-1.39 GDF15 log 17.9 <.001 26.10  5.77-118.07 ST2 log 8.7 .003 4.24 1.62-11.09 Gal3 log 6.8 .009 6.53  1.59-26.83 Endostatin 8.2 .004 88.43  4.11-1901.95 Mimecan 7.0 <.001 1.03 1.02-1.05 IGFBP7 6.4 .01 .98 .97-1.00 OPN (Osteo- 2.2 .14 1′005 1.00-1.01 pontin) Sodium 5.8 .02 .93.87-.99

All subjects with a BNP/NT-proBNP concentration >100-200 pg/mLand >1,000 pg/mL and marker/parameter levels above the cutoffs in thetable above (or below for Hemoglobin, Hematocrit, IGFBP-7, and sodium),respectively, are considered for drug therapy and/or device therapyintensification, irrespective of symptom status, perceived stability,and with careful reassessment about the presence of an “optimal” medicalprogram. Additionally, subjects with BNP/NT-proBNP concentration<100-200 pg/mL and <1,000 pg/mL, respectively and marker/parameterlevels above the cutoffs as shown above are considered for drug therapyand/or device therapy intensification. Management of HF patientsaccording to such guided combined marker guided HF therapy is the sameas with standard-of-care and encompasses all drugs, devices, andtreatment options as recommended by practice guidelines. Therapyintensification consists of increasing the dose of previously prescribeddrugs or adding drugs of a different mode of action or device therapy,exercise, diet, or a combination thereof in compliance with practiceguidelines and consistent with best clinical practices. No particularalgorithm for drug titration or drug selection is used. Although loopdiuretics may lower NT-proBNP concentrations, they are not typically beconsidered “first-line” therapy for a non-congested patient with anelevated NT-proBNP, given the lack of a mortality benefit of such agentsin the context of chronic HF.

Once medication adjustments result in the achievement of target value orthe subject is symptomatically stable, the subject is considered asbeing on a “combined marker-targeted optimal medical regimen” and istherefore removed from the few-week follow-up loop and is seen at thenext scheduled clinic visit (regular monitoring intervals). Combinedmarker levels during further visits or during near patient combinedmarker measurements (using point of care assays) may be used to furtherguide HF therapy, i.e. to further increase or reduce therapy intensityin similar visit and BNP/NT-proBNP measurements loops as describedabove.

If a subject does not meet the combined marker/parameter target goalsbut does reach a clear therapeutic limit, the subject will be removedfrom the few-week follow-up loop and will be seen at the next scheduledclinic visit. This subject is reevaluated at that scheduled clinic visitwith respect to combined marker levels and opportunities for furthertitration of medication and adjustment of treatment options. Theinvention of additional stratification according to further markers andparameters also provides benefit to patients with higher NT-proBNPtargets, e.g. 3000 pg/mL. Specifically, since not all patients can reachNT-proBNP targets ≦1,000 pg/mL, higher target cutoffs may be used and atthese levels, further markers and clinical parameters can still provideadditional risk stratification, monitoring and therapy guidancebenefits.

Compared to prior art and previous biomarker guided HF approaches, theinvention provides additional marker and parameter target levels beyondBNP/NT-proBNP target ranges. The invention also improves theidentification of patients that do not optimally benefit fromBNP/NT-proBNP-guided HF therapy.

Furthermore, associations of marker levels, therapy modifications, andoutcomes indicate that the residual risk reflected by the differentparameter above can be modified using available therapies. Thisassociation indicates that the various marker combinations can beapplied to guide heart failure therapy beyond NT-proBNP. One example ofthese therapy associations are shown below:

Combination of NT-proBNP and creatinine to guide beta blockers: Patientsguided with NT-proBNP and high creatinine concentrations at 6 monthsexperienced a good outcome with high beta blocker dose or increasingdoses of beta blockers.

Example 2 Assays

NT-proBNP was determined using Roche's electrochemiluminescence ELISAsandwich test Elecsys proBNP II STAT (Short Turn Around Time) assay. Thetest employs two monoclonal antibodies which recognize epitopes locatedin the N-terminal part (1-76) of proBNP (1-108).

IL-6 (Interleukin 6) was measured by an electrochemiluminescentimmunoassay (ECLIA, Roche Diagnostics). The test was performed using aCobas E601 analyzer from Roche Diagnostics. The test is based on a firstincubation with a biotinylated monoclonal IL-6-specific antibody and asecond incubation with a monoclonal IL-6-specific antibody labeled witha ruthenium complex and streptavidin-coated microparticles.

High-sensitive (hs) CRP was determined using a particle enhanceimmunoturbidimetric assay from Roche Diagnostics (Tina-quant CardiacC-reactive Protein (Latex) High Sensitive). In this test, Anti-CRPantibodies coupled to latex microparticles react with antigen in thesample to form an antigen/antibody complex. Following agglutination, thecomplex is measured turbidimetrically.

To determine the concentration of GDF-15 in serum and plasma samples, anElecsys prototype test was employed, using a polyclonal, GDF-15 affinitychromatography-purified, goat anti-human GDF-15 IgG antibody from R&DSystems (AF957). In each experiment, a standard curve was generated withrecombinant human GDF-15 from R&D Systems (957-GD/CF). The results withnew batches or recombinant GDF-15 protein were tested in standard plasmasamples and any deviation above 10% was corrected by introducing anadjustment factor for this assay. GDF-15 measurements in serum andplasma samples from the same patient yielded virtually identical resultsafter correction for eventual dilution factors. The detection limit ofthe assay was 200 pg/ml.

For detection of IGFBP7 in human serum or plasma, a sandwich ELISA wasused. For capture and detection of the antigen, aliquots of ananti-IGFBP7 polyclonal antibody from R&D Systems (Catalogue number: AF1334) was conjugated with biotin and digoxigenin, respectively.

Streptavidin-coated 96-well microtiter plates were incubated with 100 pibiotinylated anti-IGFBP7 polyclonal antibody for 60 min at 1 pg/ml in1×PBS solution. After incubation, plates were washed three times with1×PBS+0.02% Tween-20, blocked with PBS+1% BSA (bovine serum albumen) andthen washed again three times with 1×PBS+0.02% Tween-20. Wells were thenincubated for 1.5 h with either a serial dilution of the recombinantIGFBP7 as standard antigen or with diluted serum or plasma samples(1:50) from patients or control individuals, respectively. After bindingof IGFBP7, plates were washed three times with 1×PBS+0.02% Tween-20. Forspecific detection of bound IGFBP7, wells were incubated with 100 μl ofdigoxigenylated anti-IGFBP7 polyclonal antibody for 60 min at 1 μg/ml in1×PBS+1% BSA. Thereafter, plates were washed three times to removeunbound antibody. In a next step, wells were incubated with 75 mU/mlanti-digoxigenin-POD conjugates (Roche Diagnostics GmbH, Mannheim,Germany, Catalog No. 1633716) for 60 min in 1×PBS+1% BSA. Plates weresubsequently washed six times with the same buffer. For detection ofantigen-antibody complexes, wells were incubated with 100 μl ABTSsolution (Roche Diagnostics GmbH, Mannheim, Germany, Catalog No.11685767) and the optical density (OD) was measured after 15 min at 405and 492 nm with an ELISA reader.

Gal-3 was determined by using the BGM Galectin-3 assay (BG medicine,Waltham, Mass., USA). It quantitatively measures galectin-3 in serum orEDTA-plasma by enzyme linked immunosorbent assay (ELISA) on a microtiterplate platform. The assay utilizes two monoclonal antibodies againstgalectin-3. One rat monoclonal anti-mouse galectin-3 antibody is coatedonto the surface of the wells in a microtiter plate and serves as thecapture antibody to bind galectin-3 molecules in samples, while theother mouse monoclonal anti-human galectin-3 antibody is provided insolution and functions as the tracer antibody for detecting galectin-3molecules bound to the capture antibody.

For detection of mimecan in human serum or plasma, a sandwich ELISA wasused. For capture and detection of the antigen, aliquots of ananti-mimecan polyclonal antibody from R&D Systems (Catalogue number: AF2660) are conjugated with biotin and digoxygenin, respectively.Streptavidin-coated 96-well microtiter plates are incubated with 100 μlbiotinylated anti-mimecan polyclonal antibody for 60 min at 0.2 [mu]g/mlin Ix PBS solution. After incubation, plates are washed three times with1×PBS+0.02% Tween-20, blocked with PBS+2% BSA (bovine serum albumen) for45 min and then washed again three times with Ix PBS+0.02% Tween-20.Wells are then incubated for 1 h with 100 μl of either a serial dilutionof the recombinant mimecan as standard antigen or with diluted serum orplasma samples (1:5 in 1×PBS+1% BSA) from patients or controlindividuals, respectively. After binding of mimecan, plates are washedthree times with Ix PBS+0.02% Tween-20. For specific detection of boundmimecan, wells are incubated with 100 μl of digoxigenylated anti-mimecanpolyclonal antibody for 45 min at 0.2 μg/ml in Ix PBS+1% BSA.Thereafter, plates are washed three times to remove unbound antibody. Ina next step, wells are incubated with 100 μl of 75 mU/mlanti-digoxigenin-POD conjugates (Roche Diagnostics GmbH, Mannheim,Germany, Catalog No. 1633716) for 30 min in 1×PBS+1% BSA. Plates aresubsequently washed six times with the same washing buffer as above. Fordetection of antigen-antibody complexes, wells are incubated with 100 μlABTS solution (Roche Diagnostics GmbH, Mannheim, Germany, Catalog No.11685767) and the optical density (OD) is measured after 15 min at 405and 492 nm with an ELISA reader.

For measurement of endostatin in human serum or plasma, a commerciallyavailable sandwich ELISA (Quantikine Human Endostatin Immunoassay,Catalog Number DNSTO, R&D Systems) was used. Measurements are performedaccording to the instructions given by the manufacturer.

sST2 was determined by using the Presage™ ST2 Assay from CriticalDiagnostics (San Diego, Calif., USA). The assay is a quantitativesandwich monoclonal ELISA in a 96 well plate format for measurement ofST2 in serum or plasma. Diluted plasma was loaded into appropriate wellsin the anti-ST2 antibody coated plate and incubated for the prescribedtime. Following a series of steps where reagents are washed from theplate, and additional reagents were added and subsequently washed out,the analyte was finally detected by addition of a colorimetric reagentand the resulting signal was measured spectroscopically at 450 nm.

The biomarker mimecan was determined as described in WO2011/012268.

sFlt1 was tested using an ELECSYS immunoassay which employs twoantibodies that are specific for sFlt1. The test can be carried outautomatically using different Roche analysers including ELECSYS 2010 andcobra e411 and cobra e601.

Uric acid was determined by applying an enzymatic colorimetric method.In this enzymatic reaction, the peroxide reacts in the presence ofperoxidase (POD), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline(TOOS), and 4-aminophenazone to form a quinone-diimine dye. Theintensity of the red color formed is proportional to the uric acidconcentration and is determined photometrically.

Urea was measured by an in vitro test for the quantitative determinationof urea/urea nitrogen in human serum, plasma and urine on Roche/Hitachicobas c systems. The test can be carried out automatically usingdifferent analysers including cobas c 311 and cobas c 501/502. The assayis a kinetic assay with urease and glutamate dehydrogenase. Urea ishydrolyzed by urease to form ammonium and carbonate. In the secondreaction 2-oxoglutarate reacts with ammonium in the presence ofglutamate dehydrogenase (GLDH) and the coenzyme NADH to produceL-glutamate. In this reaction 2 moles of NADH are oxidized to NAD⁺ foreach mole of urea hydrolyzed. The rate of decrease in the NADHconcentration is directly proportional to the urea concentration in thespecimen and is measured photometrically.

Creatinine was measured creatinine was measured in plasma samples by arateblanked and compensated Jaffe method adapted for Roche/Hitachiauto-analysers (see also Foster-Swanson et al., Reference IntervalStudies of the Rate-Blanked Creatinine/Jaffe Method on BM/HitachiSystems in Six U. S. Laboratories. Clin Chem 1994; Abstract No. 361).The assay is based on a kinetic in vitro test using rate-blanking andcompensation for the quantitative determination of creatinine in humanserum, plasma and urine. Sodium hydroxide and picric acid was added tothe sample to start the formation of creatinine-picric acid complex. Inalkaline solution, creatinine forms a yellow-orange complex withpicrate. The color intensity which is directly proportional to thecreatinine concentration was measured photometrically.

D-Glucose was measured in plasma samples using an enzymatic assay fromRoche/R-Biopharm (see also Schmidt, Die enzymatische Bestimmung vonGlucose and Fructose nebeneinander, Klinische Wochenzeitschrift, 1961,39, 1244-1247). The marker was phosphorylated to D-glucose-6-phosphatein the presence of the enzyme hexokinase (HK) andadenosine-5′-triphosphate (ATP) with the simultaneous formation ofadenosine-5′-diphosphate (ATP). In the presence of the enzymeglucose-6-phosphate dehydrogenase, D-glucose-6-phosphate was oxidized toby NADP to D-gluconate phosphate with the formation of reducednicotinamide-adenine dinucleotide phosphate (NADPH). The amount of NADPHformed in this reaction is stoichiometric to the amount of D-glucose.NADPH was measured by means of light absorbance.

Plasma sodium was measured by ion-selective electrodes using plasmaspecimens by applying an Ion Selective Electrode (ISE) which makes useof the unique properties of certain membrane materials to develop anelectrical potential (electromotive force, EMF) for the measurements ofions in solution (COBAS Integra 400; Roche Diagnostics GmbH, Mannheim,Germany, Assay: “ISE indirect Na—K—Cl for Gen.2”).

Hemoglobin (Hb) was measured using the Reflotron® Hemoglobin assay. Thetest is based on the oxidation of hemoglobin to methemoglobin bypotassium hexacyanoferrate (III) (Fe2+ to Fe3+). The hemoglobin level isproportional to the color intensity and were measured at a wavelength of567 nm and 37° C.

HbA1c (glycated hemoglobin, Glycohemoglobin) was measured by using aRoche in vitro test which allows for the quantitative determination ofHbA1c on Roche/Hitachi cobas c systems (Assay: “Tina-quant HemoglobinA1c Gen.3”, Roche Diagnostics GmbH, Mannheim, Germany).

Prealbumin was measured by using a Roche in vitro test which allows forthe quantitative determination of prealbumin in human samples onRoche/Hitachi cobas c systems (ACN 710, ACN 8710). The assay is animmunoturbidimetric assay. Human prealbumin forms a precipitate with aspecific antiserum which is determined turbidimetrically.

Cystatin C was measured by using an immunoturbidimetric assay for thequantitative in vitro determination of cystatin C in human serum andplasma on Roche automated clinical chemistry analyzers (Assay:Tina-quant Cystatin C, Roche Diagnostics GmbH, Mannheim, Germany). Inthis assay human cystatin C agglutinates with latex particles coatedwith anti-cystatin C antibodies. The aggregate is determinedturbidimetrically at 546 nm.

CONCLUSIONS

The combination of NT-proBNP or BNP with other markers and clinicalparameters can be used for monitoring purposes and as a guide fortherapy in addition to current standard-of-care to adjust and titratetherapy in HF patients (chronic or acute HF after stabilization),preferably in those patients in whom HF is due to impaired systolicfunction. These markers and parameters are preferably Creatinine, eGFR(calculated from Creatinine levels), BUN, Glucose, HbA1c, hsCRP,Cystatin C, IL-6, Prealbumin, sFLt-1, Uric Acid, GFD-15, sST2,Galectin-3, Endostatin, Mimecan, IGFBP-7, Osteopontin, Sodium,Hemoglobin, and Hematocrit, as well as heart rate and QRS duration.Specifically, addition of these measurements to NT-proBNP or BNPtogether with current standard-of-care are able to further risk stratifyHF patients who are already guided by NT-proBNP but may be in need formore intensified therapy and closer observation. Thus, the presentinvention optimizes heart failure therapy guidance beyond NT-proBNP byconsidering combinations of natriuretic peptides with other markersand/or clinical parameters.

1. A method for identifying a patient who is eligible to anintensification of heart failure therapy, said method comprising thesteps of (a) measuring the level of at least one marker selected fromthe group consisting of creatinine, urea, sodium, glucose, HbA1c(glycated hemoglobin), hemoglobin, and hematocrit in a sample from apatient who has heart failure and who receives BNP-type peptide guidedheart failure therapy, and (b) comparing the level (or levels) of themarker (or markers) measured in (a) to a reference level (or referencelevels).
 2. The method according to claim 1, further comprising step (c)of identifying a patient who is eligible to an intensification of heartfailure therapy, or not.
 3. The method of claim 1, wherein the patientdisplays a level of a BNP-type peptide which is below the referencelevel for said BNP-type peptide being indicative of intensification ofheart failure therapy.
 4. The method of claim 1, wherein i) the at leastone marker is selected from the group consisting of creatinine, urea,glucose, and HbA1c (glycated hemoglobin), and wherein a level (orlevels) of the at least one marker in the sample from the patient whichis (are) above the reference level (reference levels) for said marker(markers) indicates that the patient is eligible to intensification ofheart failure therapy, and/or wherein a level (or levels) of the atleast one marker in the sample from the patient which is below thereference level (reference levels) for said marker (markers) indicate(s)that the patient is not eligible to intensification of heart failuretherapy, and/or ii) the at least one marker is selected from the groupconsisting of sodium, hemoglobin, and hematocrit, and wherein a level(or levels) of the at least one marker in the sample from the patientwhich is (are) below the reference level (reference levels) for saidmarker (markers) indicate(s) that the patient is eligible tointensification of heart failure therapy, and/or wherein a level(levels) of the at least one marker in the sample from the patient whichis above the reference level (reference levels) for said marker(markers) indicate(s) that the patient is not eligible tointensification of heart failure therapy.
 5. A method for identifying apatient who is eligible to an intensification of heart failure therapy,said method comprising the steps of (a) measuring the level of aBNP-type peptide in a sample from a patient who has heart failure andwho receives BNP-type peptide guided heart failure therapy; (b)measuring the level of at least one marker selected from the groupconsisting of creatinine, urea, sodium, glucose, HbA1c (glycatedhemoglobin), hemoglobin, and hematocrit, in a sample from the patient,(c) comparing the level of the BNP-type peptide measured in (a) to areference level (or reference levels), and (d) comparing the level (orlevels) of the at least one marker measured in (b) to a reference level(or reference levels).
 6. The method of claim 5, wherein i) the at leastone marker is selected from the group consisting of creatinine, urea,glucose, and HbA1c (glycated hemoglobin), and wherein (a) a level of theat least one marker in the sample from the patient which is above thereference level for said marker, and a level of BNP-type peptide whichis above the reference level for said BNP-type peptide is indicative fora patient who is eligible to intensification of heart failure therapy,(b) a level of the at least one marker in the sample from the patientwhich is above the reference level for said marker, and a level of saidBNP-type peptide which is below the reference level for said BNP-typepeptide is indicative for a patient who is eligible to intensificationof heart failure therapy, (c) a level of the at least one marker in thesample from the patient which is below the reference level for saidmarker, and a level of said BNP-type peptide which is above thereference level for said BNP-type peptide is indicative for a patientwho is eligible to intensification of heart failure therapy, and/or (d)a level of the at least one marker in the sample from the patient whichis below the reference level for said marker, and a level of saidBNP-type peptide which is below the reference level for said BNP-typepeptide is indicative for a patient who is not eligible tointensification of heart failure therapy, and/or ii) the at least onemarker is selected from the group consisting of sodium, hemoglobin, andhematocrit, and wherein (a) a level of the at least one marker in thesample from the patient which is below the reference level for saidmarker, and a level of said BNP-type peptide which is above thereference level for said BNP-type peptide is indicative for a patientwho is eligible to intensification of heart failure therapy, (b) a levelof the at least one marker in the sample from the patient which is belowthe reference level for said marker, and a level of said BNP-typepeptide which is below the reference level for said BNP-type peptide isindicative for a patient who is eligible to intensification of heartfailure therapy, (c) a level of the at least one marker in the samplefrom the patient which is above the reference level for said marker, anda level of said BNP-type peptide which is above the reference level forsaid BNP-type peptide is indicative for a patient who is eligible tointensification of heart failure therapy, and/or (d) a level of the atleast one marker in the sample from the patient which is above thereference level for said marker, and a level of said BNP-type peptidewhich is below the reference level for said BNP-type peptide isindicative for a patient who is not eligible to intensification of heartfailure therapy.
 7. A method for optimizing BNP-type peptide guidedheart failure therapy, said method comprising the steps of (a) measuringthe level of at least one marker selected from the group consisting ofcreatinine, urea, sodium, glucose, HbA1c (glycated hemoglobin),hemoglobin, and hematocrit in a sample from a patient who has heartfailure and who receives BNP-type peptide guided therapy, and (b)comparing the level (or levels) of the marker (or markers) measured in(a) to a reference level (or reference levels), thereby optimizingBNP-type peptide guided therapy.
 8. A method for predicting the risk ofa patient who has heart failure and who receives BNP-type peptide guidedheart failure therapy to suffer from cardiac decompensation,hospitalization, and/or death said method comprising the steps of (a)measuring the level of at least one marker selected from the groupconsisting of creatinine, urea, sodium, glucose, HbA1c (glycatedhemoglobin), hemoglobin, and hematocrit in a sample from a patient whohas heart failure and who receives BNP-type peptide guided heart failuretherapy, and (b) comparing the level (or levels) of the marker (ormarkers) measured in (a) to a reference level (or reference levels). 9.The method of claim 1, wherein the patient has heart failure classifiedas stage B or C according to the ACC/AHA classification, and/or whereinthe patient has heart failure according to class II or III of the NYHAclassification.
 10. The method of claim 1, wherein the sample is ablood, serum or plasma sample, and/or wherein the patient is human. 11.The method of claim 1, wherein the heart failure therapy is medicinalheart failure therapy that comprises administration of at least onemedicament selected from the group consisting of diuretics, angiotensinconverting enzyme inhibitors, angiotensin II receptor blockers, betablockers and aldosterone antagonists.
 12. The method of claim 11,wherein the heart failure therapy comprises combined administration of abeta blocker and an ACE inhibitor.
 13. The method of claim 1, whereinthe intensification of heart failure therapy comprises increasing thedosage of previously administered medicaments, the administration of afurther medicament (or medicaments), device therapy, life style changes,and combinations thereof.